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Team Members
If you want to learn more about us, just click the names!
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Büşra Güleçer
Name: Büşra Surname: Güleçer
Age: 16
Hometown: Elazığ, Turkey
Purpose in joining iGEM: To gain an experience about synthetic biology, designing a website and meeting with students like me from different countries
Job of your dreams: I want to be a doctor. Particularly, a heart surgeon
Plan for future: I am not sure, but after being a doctor, I may be an academician in a university and that moment I may treat poor people
Favourite Quote: Work for this world like you never die, work for other world like you will die tomorrow (Hz. Mohammad)
Favourite Music: Pop, R&B, verbal jazz, blues, and some of slow or classic songs
Favourite Movie: Millionaire
Country where you want to go: Countries like Haiti or Zimbabwe which are very poor. Maybe Palestine as there is violence. I live in good conditions. I should see them, and be grateful to God.
Personal Description: I like learning new things. So, I read, watch documentary. I like reading magazines about history, science. Also, I like recognizing other cultures, languages and religions. Especially, I read books about Islam and learn French and Arabic except for English. I am interested in NBA, but I don't follow matches,now. My favorite team is Boston Celtics and player is Rajon Rondo. I like doing tezhip( kind of Turkish art).
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Dilara Soylu
Name: Dilara Surname: Soylu
Age: 16
Hometown: Kayseri, Turkey
Purpose in joining iGEM: iGEM is a great way of understanding synbio world which is quite similar to the language of computers.
Job of your dreams: Computer engineer, physicist
Plan for future: to be helpfull to society/ to be happy/ to read 10.000 thick books
Favourite Quote: The best way to make dreams true, is to wakeup
Favourite Music: Classical music, Alternative Rock
Favourite Movie: V for Vandetta
Country where you want to go:Italy, it would be amazing to see one of the first center of science and philosophy
Personal Description: Someone trying to find out the meaning of "true", others can say waste of time but loves every issue about music, want to live in a world that everyone can pick out what to learn; without any pressure, wishing to be a masterCoder on her own, this is all how I describe myself.
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Mariye Erol
Name: Mariye Surname: Erol
Age: 16
Hometown: Yozgat, Turkey
Purpose in joining iGEM: iGEM is the way to gain an experience about synbio. Also to meet students like me from different countries
Job of your dreams: Surgeon, biotechnologist
Plan for future: I want to travel whole world
Favourite Quote: People of tomorrow shouldn’t linger with today
Favourite Music: Classical music, Pop
Favourite Movie: Dead Poets Society
Country where you want to go:It doesn’t matter for me because I would love to meet different kind of people, travel through different places
Personal Description: Hey guys, what up? Let me talk about myself. I love painting and I am interested in the calligraphy. I like playing table tennis and also playing the flute. I’m not Professional about this but it is a good way to spend my free time. Whatever sleeping is the best way to spend free time. :) that’s all :)
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Sinem Fırat
Name: Sinem Surname: Fırat
Age: 15
Hometown: Malatya, Turkey
Job of your dreams: General Surgeon
Plan for future: to be a normal person
Favourite Quote: People don't change
Favourite Music: Classical music, Pop
Favourite Movie: Harry Potter
Country where you want to go:It is Australia because I like Aussie people
Personal Description: Hey guys this is Sinem!! I don't know what am I gonna say but I'll try :) I really love playing basketball and volleyball. Also I like listening to music and some people say that I can sing :)I think I'm a cheerful person but sometimes I fell weird and I don't know why :) I love pandas and koalas, they make me feel happy. I think living your life is a great thing. All I wanted to say is, I am living my life so this is your life and just try to be happy :)
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Hatice Kurşun
Name: Hatice Surname: Kurşun
Age: 16
Hometown: Mersin, Turkey
Purpose in joining iGEM: To add knowledge to my knowledge
Plan for future: To be a beneficial person to society
Favourite Quote: In order to succeed, your desire for success should be greater than your fear of failure (Bill Cosby)
Favourite Music: Classical, Slow music
Favourite Movie: Inception
Country where you want to go:India, because you have a facility to meet people from variously different countries
Personal Description:Hi I'm Hatice. I'm glad you to meet you. I like meeting different people. I like swimming, travelling to different places. I'm interested in music, I like playing the guitar. I can't live without a sea but unfortunately I live in Ankara and there isn't any sea in here. I'm a trustworthy, cheerful and a little bit shy person. How about you people?
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Ayşe Demirci
Name: Ayşe Surname: Demirci
Age: 23
Hometown: Kastamonu, Turkey
Purpose in joining iGEM: iGEM is the port to the science world for me. I participated last year for the first time at the Collegiate Division and it was great! Now I want to assist NGSS Turkey by sharing my experiences about iGEM
Job of your dreams: A neurologist (for now :- ))
Plan for future: To be a good doctor and scientist /make a contribution to the understanding of the miraculous human body
Favourite Quote: Be the change that you wish to see in the world (Mahatma Gandhi)
Favourite Music: Actually I like EVERY kind of music;-) it makes no difference. I'm happy to be able to listen to just the right music I need for the mood I am in...
Favourite Movie: 3 Idiots
Country where you want to go:: Ireland. The nature of Ireland fascinates me since my childhood
Hobbies: Playing the violin, the guitar and the Ney (a Turkish reed flute). Basketball and football
Personal Description: I was born and grown up in Germany. Since 2011 I'm studying medicine at Turgut Ozal University. I'm a passionate violinist and I like almost all kinds of music. I joined the iGEM family last year at the collegiate division. It is a pleasure for me to advise NGSS Turkey. Thank you for the great time we had together:)
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Fatma Betül Çevik
Name: Fatma Betül Surname: Çevik
Age: 20
Hometown: Tokat, Turkey
Plan for future:to be a physician scientist
Favourite Music: Country, Classical
Favourite Movie: Hugo Cabret
Country where you want to go:: Japan, I want to observe and learn their culture
Personal Description: I’ll be third year medical student. I like my lessons without exceptions. I get excited by learning new things, especially in branches of science. I guess that’s why I take so much pleasure of iGEM studies (researches, experiments etc.). Furthermore, I think youths will achieve such incredible points in SynBio and in science as long as they are encouraged. I will try to be together with those young scientists which work with endless desire in every opportunity.
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Team Overview
Hi there,
Our team consists of 6 high school, 3 university students and 2 instructors. We are all so excited about iGEM HS 2013. We were ordinary people, who were just dealing with daily issues. Thank to SynBio and iGEM for giving such chance to us. Below you can find our team video prepared for iGEM 2013. "iGEM" brings us together"
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Photo Communication - Abstract
Communication between bacteria is a very commonly used phenomenon . It is a system which is continuously tried to enhanced and developed. As an alternative to these mostly AHL/inducible promoter dependent communicaiton systems we designed a pathway that targets messaging between different kinds of bacteria and especially bacteria in different and independent media. In our project we are going to examine the ability of a bacterium to affect other colonies via light/color production and emission as a product of several biochemical activities and additionally the effectiveness for different colors and wavelenghts.
Furthermore, for the safety part of the project, we will analyze the capability of colonies to induce the death of other colonies or activating a second antibiotic resistance via ‘’photocommunication’’ additionally to the already integrated one in the backbone against contamination risks.
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Murder Communication - Abstract
As a result of rising number of studies in the field of SynBio the role of genetically modified organisms in our lives increased and gained an important role. At the same time, to keep control of the interaction we get with them and to take precautions against possible dangers, various safety modules were developed. We also established safety system with the help of quorum sensing in our project which is focused on bacterial interactions. At this point of idea, we used LasI and LasR; the communication chemicals of Pseudomonas Aeruginosa. Response of the receiver cell will be the output of holin-endolysin enzymes to the interaction with LasI gene which will be produced by sender cell. These enzymes will cause the perforation of bacterium cell membrane system and this will result with bacterium death. Consequently, a controlled cell death pathway will be developed with the system of an inducer connected to the sender.
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Abstract
Module 1 - PhotoCommunication Abstract
Communication between bacteria is a very commonly used ... Read More
Module 2 - MurderCommunication Abstract
As a result of rising number of studies in the field of SynBio ... Read More
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Results
Module 1 - PhotoCommunication Abstract
PhotoCommunication - Result Read More
Module 2 - MurderCommunication Abstract
MurderCommunication - Result Read More
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PhotoCommunication - Result
We have started with cloning the both genes for our communication system which depends on Luciferase and Cph8. We went on two ways to do this. We did the PCRs of the primers which were designed for the genes in iGEM distribution kit. In the meantime, we did the transformation of them into E.Coli TOP10 strain.
We completed the cloning of the Luciferase system by using the parts BBa_K325219 (Red Luc.), BBa_K325108 (EPIC Firefly Luc.) and BBa_K325909 (Lux Operon) submitted by the Cambridge Team 2010; to make the sender system. For the continuation of experiments, we waited for CpH8 results.
To establish the response system which is related to the CpH8 induced pOmpC promoter in the receiver system of PhotoCommunication,; we decided to use two different assembly methods for Luciferase(BBa_K325108) and GFP(BBa_K769001). These were Gibson and 3A Assembly methods. The system we aimed to build was Cph8+pOmpC+Luciferase/GFP. Regarding the fact that we have to combine this triple with the pSCB1C3 backbone after their successful ligation, we decided to continue with Gibson Assembly. We didn’t have experience before with using the Gibson Assembly method thus, we designed primers just for one system and ordered it. We were thinking of using Gibson Assembly on the other systems according to the results of the first trials. The Gibson Assembly master mix kit was sent before by iGEM high school teams sponsor NEB.
Before we had Gibson Assembly primers, we tried to multiply Cph8, pOmpC, EPIC Luc. and GFP device systems via transformation and PCR procedures. We were thinking to do the 3A Assembly in this way. We obtained all genes with PCR except for Cph8 and EPIC Luc. because of their extended length (Figure 1). We continued with pOmpC, GFP Device and pLac attained from PCR samples. At the end of the transformation oof these parts, isolation digestion followed in order to obtain EPIC Luc. and we passed on to the 3A assembly of EPIC Luc. and pOmpC.
pLacI(9): 220 bp, Cph8(8): 4333 bp, EPIC: 2626 bp, pOmpC(6): 108 bp, pOmpC’li GFP Device(3): 990 bp.9, PCRs of 3 and 6 are done
BBa_K769001: 990 bp. We continue with the 3 and 3(2).
For 3A Assembly we needed first to ligate pOmpC and EPIC Luciferase. For this, we did the ligation to pSB1C3 from the sections of isolation and PCR purification samples . In the meantime, we tried to combine EPIC Luc. with pLac to show its working. Likewise for that combination we used isolation and PCR samples. To sum up, there were successful results for the ligation of the pOmpC promoter and EPIC Luc. which we called ‘’6’’, but the ligation of pLac promoter and EPIC Luc. again which we called this time ‘’9’’, can hardly be regarded as a success.
pOmpC(6): 108 bp, EPIC Luc: 2626 bp, pLac(9): 220 bp. pOmpC combined with EPIC Luciferase
After pOmpC and EPIC Luc. were combined, the GFP system with pOmpC was restricted appropriately and became ready to ligation, we focused on the CpH8’s cloning to add it to the top of the system.
We tried transformation again after decreasing the antibiotic concentration in the plates. There were few colonies in some plates while some of them didn’t have any colonies. We grew those colonies in decreased antibiotic liquid cultures. Unfortunately, the digestion results did not give the amount we wanted and we thought they could be the product of contamination of colonies.
We repeated transformation by taking NEB10 compotent cells which were confirmed by AUC_Turkey before, to see if the problem is about our compotent cells . The result did not change and there still weren’t any colonies.
By the way, our Gibson Assembly primers have arrived. We thought we could reproduce Cph8 in that way so we have started PCR procedure. Primarily, we did PCR on Cph8 and EPIC Luc. with long PCR enzyme system because of their extended length. We could not get any results from both of these elements (Figure 4).We did PCR for EPIC Luc. and CpH8’s with High Fidelity, too. This time, we were able to reproduce EPIC Luc. (Figure 5). In the meantime, we were doing PCR of CpH8 from iGEM BioBrick kit because we were not able to reproduce it before. Our kit was not enough to do such numbers of PCRs and transformations so we diluted this plasmid from 2013 AUC_Turkey and 2012 Fatih Medical teams kits next to ours. We did the long PCR of CpH8 with Golden Blocked PCR hoping for efficiency increase (Figure 6). Again there was no band on the electrophoresis gel after PCR.
1C3: pSB1C3(2070 bp)
In this PCR, although EPIC was reproduced , Cph8 couldn't obtained
Result of Cph8 Long PCR in golden block and Result of pOmpC Gibson Primerlers Gradient PCR
We did electrophoresis of restricted and unrestricted CpH8 on % 0.8 gel and 75V for 1 h to control the presence of gene in the matter we diluted. Again we did not see any band on the electrophoresis gel
M30109 Cph8 sistemi çoğaltılamayınca parçalardan bütüne gitme amacıyla HO1&PcyA, r8 pcr yapıldı. Aynı zamanda kitte bulunan Cph8'in LacZ'li halinin(8z) de pcr ı yapıldı.8z->BBa_K322327: 4027 bp, r8->BBa_K322124: 2253 bp, HO1->BBa_K098010: 1531 bp.
We have optimized our transformation protocol after consult to Turgut Ozal University Medical School Medical Genetics Department professors and transformed CpH8 again with a new protocol, but we still could not get any colonies. Although our 14 day and night reproduce working, we could not get any results for CpH8 so we gave up trying this system and started to wait for the delivery of our designed CpH8 device which includes Ampicillin resistance.
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MurderCommunication - Result
In our mass killing system which depends on the quorum sensing, firstly, we started with controlling the genes we got from Team Fatih Medical 2012. We did the measurement and digestion of the isolation samples of Fatih Medical. We also did digestion of the PCR purification samples and controlled them
BBa_K772005, 8:BBa_K772004
We thought to build an IPTG controlled sender system by adding a constitutive promoter at the downstream of the LasI system. We did PCR of BBa_J23100 and transformation for the same part, alternatively we tried pTetR (BBa_R0040) and BBa_J23101, too. We tried to combine these promoter alternatives and our LasI system with 3A Assembly but we could not get successful result. Whereupon we decided to reproduce and try BBa_K084007 pLac bound LasI system of Team Chiba 2008 which was added to the distribution kit and BBa_I0407 LasI series which includes ecfp under pTetR. In the meantime, we controlled the functionality of LasR-T4 Lysis system by using the LasI series (K772005) of Team Fatih Medical 2012 without adding promoter to the downstream region of LacI and by using it as a device producing LasI under the control of pLac.
To sum up, with a chemical (IPTG) inducible quorum sensing, it is possible to kill whole receiver cells in desired time.
In the meantime BBa_J23100 (5) and BBa_K772005 (7) are ligated successfully (57). LasR device BBa_K772004 (9) and T4 Lysis Device BBa_K112808 are also ligated with success.
As a result, we completed our composite parts; confirmation experiments are continuing.
Ligation Results of K990002 and K990005
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PhotoCommunication - Assembly/Design
For the installation of an interaction system which is independent from any chemicals or the medium, we used the photoreceptor called Cph8 in the literature[3]. This receptor was first used by Team UT Austin in 2004. In order to harvest the molecule PCB which is one of the most important members of the Cph8 pathway, we added to our final part the HO1 (heme oxygenase) and PcyA (phycocyanobilin=ferredoxin oxireductase) sequences which are responsible for the synthesis of PCB. As PCB molecule gets in contact with light and induces the Cph8-complex, the inner cell element of the complex, EnvZ, activates the OmpR protein by phosphorylation. The phosphorylated OmpR induces hereupon the pOmpC promoter and the genes in the upstream region can be translated.
3. ^ Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, Davidson EA, Scouras A, Ellington AD, Marcotte EM, Voigt CA (2005). "Synthetic biology: engineering Escherichia coli to see light".Nature 438 (7067): 441–2. doi:10.1038/nature04405. PMID 16306980.
In this way, we can use extracellular light to regulate intracellular events. In order to test the intercellular applicability of this regulable procedure, we built system (A). System (B) was built to analyze the practicability of the resistance gain in the safety field and other fields besides the light controllable backbone during the life cycle of bacteria.
System A:
As a sender system we need a colony which is capable of producing a light stimulus as the basic element of the communication. Because the Cph8 system works best at 660 nm(red light) we transformed our sender cells witht he red luciferase part(BBa_K325219) designed by Team Cambridge 2010.
To get concrete results from the receiver cell which will respond to the photostimulus, it was a safe way to use proteins which are simply visible through their color. In the upstream region of the OmpR inducible pOmpC promoter we placed:
a. EPIC Luciferase(BBa_K325100) ya da
b. GFP(BBa_K769001)
and intended to test the GFP or EPIC Luciferase respond of the receiver bacteria in case of a Red Luciferase stimulus from the sender bacteria.
System (B):
Here, our goal was to find out whether it is possible to strengthen the bacterial antibiotic resistance in the presence of light or - another possibility – whether we could terminate the bacterial activity of the receiver at a desired time by disabling the light source.
This was established by setting the second antibiotic resistance gene upstream of the pOmpC promoter in the receiver cell containing Cph8. In this way, if we expose the receiver to light (~660 nm) it becomes resistant to Ampicillin in our case (in addition to the Chl resistance contained in pSB1C3 plasmid backbone) and the risk of contamination by undesired colonies will be eliminated. On the other hand, if we stop the exposure our modified bacteria won’t be resistant anymore to Ampicillin and will go down with the other ones because of the still remaining Ampicillin in the medium.
Our Parts
pOmpC+Epıc
BBa_K990001
HO1+PcyA+pOmpC+Amp Res.
BBa_K990003
Cph8 System
BBa_K990004
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Data Page
Module 1 - PhotoCommunication Data
Data Page for PhotoCommunication ... Read More
Module 2 - MurderCommunication Data
Data Page for MurderCommunication ... Read More
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Module1 : PhotoCommunication - Data Page
607 nm light expands from sender cell with the help of red luciferase gene.
Through the Cph8 system, haem oxygenase and phycocyanobilin: ferredoxin oxidoreductase enzymes get activated and PCB synthesis begins. Light activated PCB induces Cph1 and histidin kinase effect of EnvZ gets activated. OmpR gets phosphorylated with the effect of Kinase. Phosphorylated OmpR alerts and activates pOmpC. With the operation of Cph( system, as a response to red luciferas stimulant:) the response is recieved at bright light with EPIC luciferase
the response is recieved with GFP
Amp resistance also gets activated
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Module2 : MurderCommunication - Data Page
Under constitutive promoter, LacI synthesis represses pLac working.
LacI influence represses by adding IPTG to the medium and in our sender cells LasI produce starts and spreads.
In receiver cells, under control of constitutive promoter, lasR is produced constantly
The LasI's connects with LasR molecules in receiver cells and activates pLas.
Holin and endolysin reproduces by reading T4 lysis device.
Endolysin drills from an outside layer so the bacterium begins to die.
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At a Glance
Photo Communication - At A Glance
Our project consists mainly of two modules. These modules basically depend on the communication between bacteria. Our main target was communication via light without the need of chemicals as intermediate agents. Besides this, we have also built a collective killing system which is controllable with chemical inducers.
In our PhotoCommunication module, we used the Luciferase system, the bioluminescence genes from fireflies which were added to the partsregistry in 2010 by Team Cambridge. Regarding the fact that our receiver device works best at around 660 nm wavelength which represents red light, we installed Red Luciferase into our sender cells (BBa_K325219).
In the head member of the PhotoCommunication module – the receiver bacteria – we applied the Cph8 photosensitive device (BBa_M30109) as photoreceptor which was submitted in the partsregistry by Team UT_Austin in 2004. In this system, membrane proteins PCB and Cph1 interact in the case of light exposure. As a result the histidine kinase enzyme activity of another membrane protein EnvZ gets activated and phosphorylates the endogenous transcription factor OmpR. Hereupon, OmpR induces pOmpC promoter and right after light/Cph8 activation the promoter activity of pompC starts.
We built several systems in the PhotoCommunication module for different feedbacks of the receiver realted to the stimulus:
1) The Luciferase Respond: The receiver induced by the red light from the sender, answers with the blue light which is produced by the Luciferase translated under the activated pOmpC promoter. This will be the visual proof of bacterial communication.
2) The GFP Respond: This time, the receiver answers by producing green fluorescent protein (GFP) instead of the Luciferase system.
3) Control of Antibiotic Resistance: The purpose here is that colonies show a second antibiotic resistance in a bright environment (appropriate wavelength is required) in addition to that one which is already integrated in the backbone due to the sequence coding for Ampicillin-resistance under the pOmpC promoter. On the contrary, the same colonies shall lose their extra antibiotic resistance in case of interruption of the light stimulus.
Murder Communication - At A Glance
In module 2, we did a communication system which depends on quorum sensing. For this, we used LasI-LasR which is a communication chemical of the gram(-) bacteria called P. Aeruginos. This system works with a similar to interaction between LuxI and LuxR mechanism. The LasI produced by sender gets out of the cell and connects with LasR in receiver cells.The LasI-R complex activates pLas promoter and the sender controlled pathway in receiver cells starts working. Thus, we decided to use this system to kill the colonies which switch on their reciever mode at any time we want. We added T4 lysis device after pLas in reciever cells. T4 lysis device; provides the puncture of the cell from inside with holin enzyme and from outside layer with the endolysin enzyme. Thus, LasI coming from sender activates the pathway that will kill the receiver cell by ensuring the rupture of the cell membrane of the receiver bacteria.
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Murder Communication - Assembly/Design
In view of the enormous problems we had with the cloning of Cph8 parts and the fact that our self-designed Cph8 based genes will arrive much later than expected, we decided to develop an alternative project to PhotoCommunication. We started with thinking whether there was a way to make a new project based on our knowledge from the research on bacterial communication methods. As a result of our brain stormings, we came to the result that we want to establish a killer system and link it to the quorum sensing pathway in our former project ideas.
There was the option for us to use the optimized quorum sensing LasI/R genes of Team Fatih Medical 2012. Finally, we used the LasI/R quorum sensing mechanism of the gram(-) P.Aeruginosa and combined IPTG-inducible LasI production in the sender bacteria with the LasI/LasR dependent T4 Lysis device in the receiver bacteria in order to induce the death of the receiver.
We used here for the sender BBa_K772005 from Team Fatih Medical 2012. In order to be able to control this device with LacI and IPTG, we decided to add a constitutive promoter at the downstream region of LacI. For this purpose, we ligated part BBa_K772005 separately with these promoters: BBa_J23100, pTetR and BBa_J23101. In this way, we got a sender which produces LacI constitutively and inhibits the transcription of LasI at the upstream region of by repression of pLac. After adding IPTG, the inhibitory effect of LacI on pLac will disappear and the production of LasI can start.
For the receiver we also used a part from Team Fatih Medical 2012: BBa_K772004.Las Receiver Device. This time, the constitutive promoter pTetR is at the downstream of the LasR gene and ensures the constant production of LasR so that it can interact any time if a LasI molecule from the sender arrives. After interaction, LasI/LasR complex activates pLas promoter and thereby the translation of the upstream genes. At this point, we chose T4 Lysis Device which will be responsible for the bacterial cell death and worked with BBa_K112808 designed by Team UC Berkeley 2008. In conclusion, we have built a quorum sensing inducible lysis system by joining these two parts.
Our Parts
IPTG Inducible LasI Device
BBa_K990002
LasR Device+T4 Lysis
BBa_K990005
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Assembly/Design
Module 1 - PhotoCommunication Assembly/Design
For the installation of an interaction system which is independent ... Read More
Module 2 - MurderCommunication Assembly/Design
In view of the enormous problems we had with the cloning of Cph8 parts ... Read More
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Safety
1. Would any of your project ideas raise safety issues in terms of:
a. Researcher safety:
Actually few hazardous chemicals and solutions such as EtBr are used in some lab procedures such as gel preparation and electrophoresis. However these chemicals and solutions are used according to the safety rules of the laboratory with care and caution. All the members were trained for safety regulations of the laboratory as well as toxicity of the chemicals and solutions before starting the current project.
b. Public Safety:
When released by accident, our parts and materials actually cause no damage to the general public. Due to the unability of E.coli strains, TOP10 and BL21 to survive out of the lab, they cannot pose any risk to the safety and health of the general public.
c. Environment:
E. Coli strains TOP10 and BL1 have very limited ability to survive outside the laboratory; so that, it would be unable to survive or disseminate. Therefore, there is no specific environmental risk associated with the E. coli strains. All bacterial wastes are kept in 10% bleaching solution for one day, and then, are autoclaved to be sterilized. Yet, undesired GMOs may achieve ecologically harmful features.
2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?
Solely, possible GMOs (genetically modified organisms) which are produced unwillingly during the lab procedures may cause unforeseen and incalculable consequences.A specific part of our Project ‘’MurderCommunication’’ is based on the prevention of GDOs. It contains quorum sensing dependent bacterial lysis. The cell wall destruction system consists of endolysin and holin proteins which are responsible for the lysis of the bacterium cell by degrading its cell wall.
3. Is there a local biosafety group, committee, or review board at your institution?
The institution which we are using for laboratory facilities has its own biosafety rules. Rules for laboratory use, general principles, prevention from hazardous materials and application of emergency intervention in case of accident are included. The web link is given as (http://www.fatihmed.edu.tr/icerik/guvenlikkilavuzu.php). In Turgut Ozal University Medical School, Laboratory and Patient-Employee Safety Committee is responsible for control as well as biosafety of laboratories and safety of patients and employees. This committee works under one of the vice medical director of Turgut Ozal University Hospital, Prof. Dr. Mehmet Gunduz. Form on safety rules of Turgut Ozal University Medical School Laboratory use was filled in as required. We discussed our project with Prof. Dr. Mehmet Gunduz. Safety and security issues are found sufficient enough that no change is considered as necessary. (Further questions can be directed to Prof. Gunduz, tel: +90-312-203 5103, mgunduz@fatih.edu.tr)
Our advisor provided us with biosafety and lab training before starting our project. In the training, general safety rules of laboratory use, prevention from hazardous chemicals and solutions as well as emergency intervention in case of accident were included such as:
- Eating, drinking, storing food and smoking are absolutely not allowed.
- Mouth pipetting is not allowed; modern pipettes are used in lab.
- Hazardous wastes and ordinary wastes are separated and cautiously disposed.
- Lab coats are obligatory to wear in the lab and during on-going experiments.
- In electrophoresis room, lab coat, protective eyewear, lab masks and gloves have to be worn. In the case of use of EtBr; extra caution is required.
- Washing hands after any experiment and after touching anything related with viable material is obliged.
- Air conditioning is kept closed during on-going experiments in order to avoid possible infections of spores and bacteria.
In addition, we have made an instructive video about lab safety instuctions.
Turkey has national biosafety regulations and the link is given as http://www.tbbdm.gov.tr/Home/BioSafetyCouncilHome/BioSafetyCouncilHomeChoose.aspx.
4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?
- First, IGEM committee may prepare a lab safety acknowledgement form like OSHA form. This form can be required to be filled in by all members. This will allow us to confirm whether members of the teams are informed about safety issues and standardize the safety measurements.
- Second, iGEM committee may organize a webinar to inform the participants about safety and security in labs. Webinar can be done at the beginning of the experiments. Mentor scientists may talk about their experiences and mention some tricks about safety issues.
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Overview
Module 1 - PhotoCommunication Overview
The communication between bacteria is provided by chemicals like ... Read More
Module 2 - MurderCommunication Overview
Bacteria communicate among each other by using chemicals called ... Read More
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Goals
Module1:PhotoCommunication & Module2:MurderCommunication – Goals
Achievement of communication between colonies which exist in different environments:
The communication between bacteria is provided by chemicals like AHL(N-Acyl Homoserine Lactones) in gram(-) bacteria, oligopeptids in gram(+) bacteria and some other molecules. These ways of messaging is possible only if both – sender and receiver of the message – are in the same medium, while messaging between two different media is not possible which is a limiting factor. As a solution for this standing problem and an alternative quorum sensing mechanism, ‘’Photocommunication’’ provides a non-chemical and contactless way of messaging between organisms.
Achievement of communication between different types of bacterial strains:
Between some types and subtypes of bacterial strains communication is already possible by means of different chemical molecules. However, it is known that not all types of bacteria have this ability. These microorganisms need alternative ways of interacting where ‘’photocommunication’’ comes into play.
Achievement of an new controlable prevention method against contamination:
Even if we use antibiotics in order to prevent the contamination in solid or liquid growth media, it is still not 100% safe against contamination. Our purpose is to use a second different antibiotic resistance for eliminating contamination as far as possible. The bacteria which we use in our exeriments will be able to survive by activating a second resistance at bright places thanks to the Cph8 photoreceptor system while unwanted bacteria die because of the lack of resistance against the second antibiotic.
Controlled inacivation/killing of the bacteria:
They same controlable system as mentioned above can also be used for avtivation and killing bacteria at a desired time. This time, the light source which had activated the second antibiotic resistance will be disabled so that bacteria which have done their jobs will be killed because of an antibiotic in their medium which they can’t resist any more. Thus, a processes can be terminated at each phase of the project, whenever desired -and more important- all this in a clear GDO-free manner.
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PhotoCommunication - Overview
PhotoCommunication – Overview
The communication between bacteria is provided by chemicals like AHL(N-Acyl Homoserine Lactones) in gram(-) bacteria, oligopeptids in gram(+) bacteria and some other molecules. These ways of messaging is possible only if both – sender and receiver of the message – are in the same medium, while messaging between two different media is not possible which is a limiting factor.
In our project, we aimed to provide a solution for this problem and an alternative way for bacterial communication by using the light-contollable Cph8 photoreceptor system which was used quite early in the iGEM history (2004 Team UT Austin with the ‘’photographer bacteria’’) and called our project ‘’PHOTOCOMMUNICATION’’. Through the mechanism we have developed our bacteria(receiver) will be able to perceive the light that is produced by another bacteria(sender) and as an answer a further pathway in the receiver will be activated.
We prefered to work with E.coli in our project and tested two systems which will be applicable in different areas. In our first system (A), Cph8 photoreceptor was used to get an answer signal to the Cph8 induction in form of another Luciferase product or GFP. and in the second system (B) to develop an additional antibiotic resistance.
In system (A), our goal was to confirm the proper function of Cph8. The red light which was produced by the sender through the enzyme Red Luciferase activates the receiver Cph8 photoreceptor. After receiving the photons, EnvZ membrane protein which is anchored to Cph8 will phosphorylate another protein called OmpR at the inner side of the bacterial cell. Subsequently, OmpR will activate the pompC promoter and the sender bacteria will beginn to emit another luciferase product as an answer. Consequently, this would prove that the ‘’photocommunicaiton’’ mechanism has worked.
Our aim in system (B) was to use the CpH8 photoreceptor in order to earn the receiver bacteria an extra antibiotic resistance (apart from the one in the backbone) or, the other way around, to ensure the death of the receiver type bacteria by disrupting the connection at any desired time with the light/photon source so Cph8 can’t receive any activator signals any more and the bacteria loses it’s extra resistance placed behind the pOmpC promoter and is no more able to resist the second antibiotic in the medium.
The part sequences of system (A) and system (B) are identical from the pBAD promoter and pOmpC promoter. The differance between the both systems is the sequence after pOmpC. In (A) pOmpC is followed by Luciferase in 5’ direction while in (B) it is followed by the Ampicillin resistance gene.
We have shown the bacterial communication system we have designed via two different products. We think that by changing the sequence in the upstream region of the pOmpC promoter, it is possible to control divers pathways through an interbacterial communication.
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Murder Communication - Overview
Bacteria communicate among each other by using chemicals called autoinducers. As a result, features like symbiosis, virulence, motility, antibiotic production and biofilm formation are modified at the gene expression level. This type of communication – called quorum sensing – can be amoung the same colony as well as between gram(-) and gram(+) bacteria. Regarding our former work, we decided to continue with communication between bacteria and to use quorum sensing molecules produced by the gram(-) bacteria P. aeruginosa and a similiar autoinducer chemicals like LuxI/R from another gram(-) bacteria V. Fischer - LuxI/R. [1][2]
This time, we thought of a innovative lysis device, similiar to that of the safety part of ‘’photocommunication’’. Contrary to ‘’Photocommunicaiton’’, bacteria will send each other chemical inducers instead of photons.
As a conclusion, we built a killing device wich is still related to bacterial quorum sensing mechanisms but additionally inducible with IPTG. We think that IPTG induction can serve as a control during the experiments on different colonies in the same media.
1..^ a b Passador, L.; Cook, J.M., Gambello, M.J., Rust, L., and Iglewski, B.H (1993). "Expression of Pseudomonas aeruginosa virulence genes requires cell-cell communication.". Science 260: 1127–1130. doi:10.1126/science.8493556. PMID 8493556.
2.^ a b Brint, J.M.; Ohman, D.E. (1995). "Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RhlR-RhlI, another set of regulators in strain PAO1 with homology to the autoinducer responsive LuxR-LuxI family.". J. Bacteriol. 177 (24): 7155–7163. PMC 177595. PMID 8522523
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Adventures of E.coli and B.subtilis
Hi Dear iGEMers and the others,
Have you met E.Coli and B.Subtilis? Yes, these are the cute little bacteria that you are working on them at laboratory. They are small but smart bacteria. They are trying to be heard by humankind on twitter. Not just that, they are also instruct us about iGEM and synthetic biology. Of course they are doing it with their own interpretation.
Beside to be so active like this, both of the bacteria have different characteristics although they get along with each other. Let's get to know them closer:
E.Coli: Born on 1885. He is the unique king of the Eubacteria Kingdom. He is friend with B.Subtilis over a hundred year. He loves living in large intestine most. On the other hand, he wouldn't say no for a chocolate cake. His lifetime wish is to be the king of the world -or the universe with his words- when the day come that evolution has been completed.
B.Subtilis: Born on 1835. He has a modest lifestyle. E.Coli is his best friend-maybe because of he is his only friend. He hates antibiotics. Quiet places is his best. He prefers lemonade to chocolate His lifetime wish is to go to Pollyanna's village and continiue to his modest life. Although he is modest he says "When the world orders have changed, They will show the respect that I deserve!"
If you wanna learn more about E.coli and B.subtilis, now visit and follow their twitter addresses!
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Information Stand at Mall
As a human practises work, we were trying find out a influential way to inform the society about iGEM and SynBio. We decided on the group that would talk to. Public awareness was a serious issue, therefore we claimed the public as our first group.
Subsequently, we debated about how to inform the public effectively. We found out some efficient ways, and epening a stand in a shoppingmall was one of them. We choosed Nata Vega as a place, because it is the one of the biggest shoppingmall in Turkey. It also has famous brands which are only exist in there.
On 6 April 2013, our group has moved to the mall. We managed to complete our stand on 10.00 o’clock when the visitors has just reached. Our school has assists us with the bus to carry our materials. Days before, we had have sent our brochures for distrubuted the shops under the roof of NataVega.
We were ready with our balloons, brochures and ofcourse knowledge of SynBio, iGEM. We did estemeed number of presentations. Balloons were great idea to attractc people to our stand. We talked about what is SynBio and iGEM, example projects. Busra had borrowed her uncle’s camera to take professionals photos.
We did an attribution to be able to be better. Mariye and Dilara have distrubuted booklets and informed the people who visited our stand. Busra have taken photos. Beril and Sinem have distrubuted the balloons, booklets and also invited people to our stand. Most of the visitors were families, except that business men and student groups visited our stand as well.
After six hours, we have finished our presentations. It was a hard experience for us coming back with those equipments, they were really heavy, but it worth. We met people who can help us in our future projects, too. We met people who can help us in our future projects, too.
Here are the original brochures that we distrubuted on the mall (You can see the images with full size by clicking the small images)
Here are the English version of out brochures
Below you can find some photos from our mall event
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bactUNO Card Game
In order to explain our project and SynBio better, we designed a game, which consists of the parts used in our project. The game based on famous card game, UNO. We modified some rules, but they are almost the same. Here are the cards and various descriptions:
The aim of the game is to get rid of all your cards. You need to play your cards when it is your turn. You have two choices. You can play the cards, which has the same type (colour). If you have not got the card which has the same colour with the card on the ground, you can play a card which forms the systems described. It is just needed your card to be come after the right part.
If you have got the same card which is on the ground, you can play it whether it is your turn by saying “stop”.
Red cards are terminators
Green card are promoters
Yellow cards are RBSs
Blue cards are protein sequences
NGSS cards, change the colour on the ground according to user’s preferences
iGEM cards, change the cards holder according to clock way
+2 cards, If you have got a +2 card, the person comes after you have to pick up two cards from the card stock. If the ones comes after you also have got one, this person play her / his card and it becomes to one after her / his. In these cases, last person have to pick up the sum of the cards on the ground.
+4 cards, have got the same role with +2 card, but the person can change the colour who plays the card latest.
Stop cards, stop that who is after you.
Turn cards, change the way of the game.
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SynBio Day at Kindergarden
We organized a SynBio day at the kindergarden. Our purpose was teaching new, different areas to kids and endearing science. We also aimed to measure their understanding of bacteria. First idea has came with the question :
"SynBio was a science area on university level. Although İgemhs competition showed us even high school students can understand and learn specific knowledge from SynBio. Why don’t we inform kindergarden students with the amount and level of information they can be able to understand?"
Those kids going to be futures astronauts, policemen, doctors, teachers etc. Does anyone ask how properly they know being an astronaut? So they can understand what we do, what bacteria are. We contacted with the teachers in Burc Child Academies, and explained our purpose. They were tend to do these kind of activities, so they let us to spend some good time with our little friends :)
On the first Kindergarden SynBio Day there was a group of kids aged between the range 3 – 6. Hatice Kursun, Sinem Fırat and Busra Gulecer went to Burc Child Academies, on 7th June 2013, at 10.30. Our friends prepared a great presentation on Powerpoint which is really colorfull and attractive. Presentation included brilliant pictures about bacteria. They also prepared a sketch, consisting of two beneficial hand made cartoon bacteria, to make children understand better.
Here are the notable questions that our friends asked to kids before the presentation and kids’ reaction to the questions:
Have you ever heard the word bacteria?
- Most of the kids have had heard about it.
Do you know that which functions bacteria have?
- Our friends got such answers: they sicken us, harm our teeth. The word bacteria didn’t not have a good impression on them.
Our friends have informed them with their presentation. They showed kids that there are some beneficial bacteria as well, and they are useful. Finally kids decided to pick our cartoon bacteria as friends :)
The first Kindergarden SynBio day passed great. Second Kindergarden SynBio day will come with surprises !!!
Below you can find our photos
Below you can find our powerpoint slide
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Survey
Our purpose in conducting this survey was to observe if people have enough information about synthetic biology, bacteria and activities which are related to them, or not. We surveyed with over 100 people. Most of them are high school students. The survey consist of 10 questions with 3 answer options.
You can find our survey in Turkish
You can find our survey in English
Question - 1
Are all kinds of bacteria harmful?
98% of the participants think that not all kinds of bacteria are harmful and say that some bacteria are even useful. It is suprising.
Question - 2
Do you have enough information about bacteria?
Most of participant haven’t got enough information about bacteria
Question - 3
Might bacteria be useful for the humanity?
97% of the people we have asked answered "yes" to this question.
Question - 4
Do you think that transferring genes to bacteria in order to gain various useful functions is justifiable?
For %38 of participants it is not justifiable. We need to inform people more about SynBio and its works.
Question - 5
Do you know which functions bacteria have/might have (except for illness)?
That is a good ratio. Still there are some people don’t know about bacteria’s function
Question - 6
Do you think that changing genes is acceptable?
Most of the participants think that transferring genes is ok, but changing genes on the other hand is not right.
Question - 7
Have you ever heard of E.Coli?
%55 of the participants have not heard about E.coli
Question - 8
Might be bacteria enjoying?
Most of them said that bacterias might not be enjoying.
Question - 9
Do you think that you have enough knowledge about synthetic biology?
60% of the people noticed that they don’t know enough about SynBio.
Question - 10
Do you follow competitions and activities about Synthetic Biology?
82% of the pople don’t know about activities related to bacteria/SynBio like iGEM.
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SynBio Presentation
As 2013 iGEM HS participants, NGSS TURKEY, we made an Introduction to Primary School Students. On 15th of May, we invited them to our school and organized a well designed presentation. They were all senior year students at a primary school, and as they have a High School Entrance Examination (SBS), SynBio and iGEM presentation was like a breathe for them.
Beril Gurdap has presented and Busra Gulecer has taken the photos, and she also prepared the presentation. We gave place to important notes in Powerpoint, and prepared a presentation according to that. The presentation was consisting of brief descriptions and lasted for nearly 45 minutes. Beyond the PowerPoint presentation Beril also gave place to information about our team, NGSS TURKEY.
Firstly, we explained what SynBio is. We mention the differences and similarities between genetical engineering and SynBio. We measured how the audience know about SynBio. We proved again that SynBio is a young field of science.
As every slide included iGEM logo, we explained what the igem is, too We got th attention on this part. They asked about how to participate. We explained them in the way as good as we can. We picked up next years iGEMers :)
The heart of İgem, SynBio. Pipettesss!!! We could not afford one, so we could not show what it is like in real life, although our media was able to make sense. These magical tools... They are certainly amazing! After those reactions we have to explain to our visitors that they can not buy one for themselves and keep it. Actually they can, but without eppendorfs and plates what to do with pipettes?
We showed them a plate and explained the points on the plate. It is so hard to believe that on these little plates there are more than millions of bacteria. We continued with sample projects of previous years. They were impressed by the shined bacteria. When we talked about the power of this light, we gave reading a book in front of luminous bacteria as an example. We also talked about the future of SynBio and asked them what they want e.coli to do.
Seeing smiling faces made us feel good :)
Below you can find our presentations
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Introduction of SynBio to Companies
On SynBio projects, presentations of the projects are as important as what projects are. As synthetic biologists, beyond SynBio, as scientists our intention is to inform society about new technologies, about how to make the world a better place to live and to increase the awareness of public. Society sometimes refers kids or students and sometimes people in Supermarkets. There is also one more group of people. They are businessmen. Gaining the attention of those people has slightly different benefits to SynBio Society.
Our purposes on SynBio and İgem introductions to companies have similarities with our other presentations. We are trying to inform those people about the area that we are working on. Almost ;) in every attendance we take good feedbacks. People like to hear new things, especially from youngsters. Their supports are considerable for us. With their encouragement we hope one day we are going to be able to be beneficial for humanbeings.
To present SynBio and İgem we have contacted with several companies. After couple of struggles we managed to start a communication with some of them. We prepared a stable presentation including SynBio, iGEM, Example Projects and Our Team.
Energy Market Regulatory Authory
We have contacted with Osman Birgin, who is the head of Energy Market Regulatory Authory, he was enthusiastic for such kind of activities, so he has accepted our offer. We have taken appointment from him for 5th of June 2013 , at 11.00 am. Mariye Erol and Dilara Soylu as the students and Fatma Betül Çevik as the advisor have gone for meeting. When we arrived, we had a conversation in Osman Birgin’s room. Afterward we passed to meeting room and started our presentation. The presenatation has been done to Omer Birgin, other department presidents and a septet of EMRA workers.
The audience has listened the presentation of little-known discipline synthetic biology and iGEM competition carefully. We’ve informed them about the studies on İgem related to energy. Our friends have answered the questions. Then we talked about the future of SynBio. Osman Birgin offered us sending our presentation documents as an email to the ones on their email lists who are almost five hundred people.
We informed them about our outreach projects. After couple of time, Osman Birgin offered to us sending our presentation document as email to the ones on their email list who are almost five hundred people (Document was helpful :)) We have left EMRA about 12.40 am. Beyond being able to reach our purposes, we enjoyed to spend time with them!
Halıcızade Company
From our team; Mariye Erol, Dilara Soylu and Fatma Betül Çevik have established communication with the founder and the head of Halicızade Company, Metin Özer. We have talked about whether we can make a presentation or not Metin Ozer has accepted our offer.
The presentation has been placed on 5 June 2013, at 14.00. It has lasted for 30 minutes. Firstly we explained what SynBio and İgem is. Then we have showed the works of previous teams. After we completed the presentation, they spent some time on possible kinds of projects that can be done in the area of SynBio. It was a beneficial presentation for us and him, Metin Ozer has given his thanks. Afterward we left him our presentation and sponsorhip document and left Halıcızade Company at 14.00.
Below you can find some photos from the our presentations
FIRAT COMPANY
Metin Fırat has accepted our presentation offer kindly. On 18 May, at 10.00 a.m. Sinem and Hatice from our team went to Fırat Company for the presentation. Audience consisted of six person. We used the powerpoint slides to inform the listeners. After sincere conversation we left the company. It was a succesfull presentation, we also developed our presentation skills.
Below you can find some photos from the our presentations
Here are our presentation documents :
Click here for our powerpoint slide in Turkish
Click here for our powerpoint slide in English
Click here for our sponsorship and presentation document in Turkish
Click here for our sponsorship and presentation document in English
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iGEM for Everyone
The time that we have spent in public transportations, most of the time, is wasted time. Imagine that you are in the subway, in the bus, walking or shopping; group of people came out started to tell you about such intersting issues. Wouldn’t it great? Today you learn something new dude!
Why am I telling that? We are also part of the mass. In order to arouse the interest We printed some cute brochures - OK they were well designed but we couldn’t print them all in colours(By the way, we also used the last brochures which were left over from the information stand in the shopping mall ). It was our goal to make iGEM understandable for every member of the society.
Where have we made our presentations ? Subway, bus, shopping mall, street, picnic area – everywhere we could see any human beeing! If we had a sea or a river in the neighborhood ships couldn’t escape us either. We informed people about SynBio and iGEM by leaving out the technical language out as far as possible, so even children could understand us.
We had very positive feedback and especially elderly persons stated that they are very proud of us to take part in scientific projects like iGEM at such a young age.
We also have some funny moments (especially in the subway). It was a really nice experience!
Below you can find our brochures
For English
For Turkish
Below you can find our photos
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First Week of June
06.01.2013 Saturday
- • We have transformed Luciferase, Red Luciferase,EPIC, pOmpR, pLac, RFP (Amp),Rzt, pOmpR+GFP, Cph8. (Tomorrow will taken at 10.40 am)
- • We have talked to Gökhan Nas for the pirimers which are necessery for Gibson.
- • 4 plates with Amp and the whole lab has been prepared.
- • The plates wih Chl(1) and AK(5) used from which has iGEM sent.
- • For AC and Amp, we used which we have prepared.
06.02.2013 Sunday
- • Only EPIC(1),Luc(1), pLac(1), pOmpR+GFP(3), RLuc(1) colonies grew
- • We thought that the problem could be over antibiotic sowe have transformed full 9 genes with 0.5 Chl and 0.75 Amp again. (tomorrow will taken at 2.20 am)
- • 0.5 Chl and 0.75 Amp plates have prepared and mot of them used.
- • For Cph8, we did two different examples on Amp and Chl plates. The AC plates that we have are not useful.
- • For AK (Rzt), we have made two dfferent examples from 12.12 date plates and iGEM plates
- • From Saturday: EPIC, Luc, pLac, pOmpR+GFP,RLuc examples’ liquid cultures have done. The isolation will be on tomorrow with team.
06.03.2013 Monday
- • The kit of plasmid isolation is ready.
- • EPIC,3,RLoo,Loo has isolated.
- • EPIC and 3 have cut from Xp; Loo and RLoo from ES but we have failed.
- • The liquid cultures from tomorrow have isolated.(EPIC,3,6,9,Loo,8)
- pLac: 9
- Luciferase: Loo
- Red luc: RLoo
- EPIC: EPIC
- RFP: RFP
- Cph8: 8
- pOmpC: 6
- RBS+LacZ+term: π
- pOmpC+RBS+GFP: 3
- The parts that we are going to make: 9EPIC, 83, 6π, 86π
06.04.2013 Tuesday
- • PCR of 3,6,9,8 have done.
- • 8 and EPIC’s electrophoresis results are abortive.
- • Loo and RLoo transformed.
06.05.2013 Wednesday
- • 8 and EPIC PCR again. (2.30 pm)
- • 8 and EPIC electrophoreis results are abortive.
- • 8 and EPIC PCR again(4.00 pm)
- • RLoo transformation is succesful but Loo is not.
- • Loo,RLoo transformated. (we will get at 01.05 am)
- • RLoo liquid culture has done. (we will get at 00.20 am)
- • 8,EPIC,RFP transformated. (tomorrow at 8.40 am)
- • 8 and EPIC electrophoresis results are abortive again.
06.06.2013 Thursday
- • The liquid culture of Loo and RLoo which we got at 1.05 am have done.(will come out at 1.30 am)
- • The RLoo from 1.20 am have isolated.
- • 8 has transformed. (will come out at 9.00 am)
- • The digestion and electrophoresis of 3,6,9 from PCR; RLoo(1),RLoo(2), RLoo(3),EPIC(2), EPIC(3),3(2), 9(3) from isolation have done. 6,9,9(3) are unsuccesful because of the problem with gel. RLoo(1), RLoo(2), RLoo(2) are unsuccesful. 3 and 3(2) are succesful. EPIC is suspecious.
- • EPIC’s liquid culture have done.(will come out at 8.30 am)
06.07.2013 Friday
- • The electrophoresis of 6,9 from PCR and 9(3) from isolation have done. Two gels have prepared.(%1 and %2) (80 V 40 mins.)
- • %2 gel failed.
- • %1 gel is succesful; 6 and 9 are able to use.
- • The RLoo and loo from 1.30 am have isolated.
- • Loo(2), Loo(3),RLoo(1), RLoo(2) and RLoo(3) have isolated.
- • Loo(2), Loo(3),RLoo(1), RLoo(2), RLoo(3), EPIC(1),EPIC(2) and EPIC(3) have digested. EPIC(2)’s electrophoresis result is succesful but EPIC(1) and EPIC(3) ares suspecious.
- • Loo(2), Loo(3),RLoo(1), RLoo(2) and RLoo(3) are unsuccesful.
- • Loo and RLoo’s PCR have done.
- • Loo and RLoo’s PCR results’ electrophoresis have done and we have failed again.
- • Loo,RLoo,3(3),500 are digested.
- • 8’s liquid culture have done(will come out 8.00 am)
- • Loo and RLoo’s electrophoresis have failed again!
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Second Week of June
06.08.2013 Saturday
- • 8’s liquid culture have failed.
- • Loo , RLoo and 3(3)’s digestion examples’ electrophoresis have done.
- • Loo and RLoo’s bands are not on the right places.
- • 3(3) had not cut.
- • 500 is succesful.
06.09.2013 Sunday
- • The liquid cultures of 6-EPIC 1, 6-EPIC 2, 9-EPIC 1, 9EPIC 2 have done which came out 5.30 am and 7.10. (The liquid culture A will be on 20.15 and B will be on 22.45)
- • 8 has transformated.(will be on 1.10) It has done with 5 different antibiotic resistanced plates (amp, chl, AC,AK,KAN)
06.10.2013 Monday
- • The 8 plates went out at 01.15 am. (threee plates have nothing,a plate (chl) have 3 colonies and another plate (amp) have a colonie.
- • All of the 4 colonies' liquid culture have done. (8-amp, 8-chl 1, 8-chl 2, 8-chl 3)
- • A(20.15) and B(22.25) liquid cultures have isolated. The results are fine.
- • Isolated and after that digested.
- i. 6 EPIC A 2/1
- ii. 6 EPIC A 2/2
- iii. 6 EPIC B 1/1 6EPIC's- Xba1+Pst1
- iv. 6 EPIC B 1/1 9EPIC's-EcoR1+Pst1 (have cut with)
- v. 6 EPIC B 2/1
- vi. 9 EPIC A 1/1
- vii. 9 EPIC A 1/2
- viii. 9 EPIC A 2/2
- • Electrophoresis have done. The examples have walked a little.
- • Electrophoresis of the same examples have done again.
- • Two competent cells have taken from AUC TURKEY team just for trying and CpH8's and Red Firefly Luciferase's (lc325239) transformation have done. (will have at 07.15 pm)
- • RLoo and Loo's liquid cultures have put to the ETÜV little bit opened. (will be at 19.40 pm)
- • 6 EPIC A 2/1 ,6 EPIC A 2/2,6 EPIC B 1/1, 6 EPIC B 1/1, 6 EPIC B 2/1,9 EPIC A 1/1, 9 EPIC A 1/2, 9 EPIC A 2/2 have digested again.
- • The resultant RLoo and CpH8's (7.15 pm) liquid cultures have done. (will be at 11.45 am) (5 Loo, 4 RLoo, 4 CpH8)
- • Loo, RLoo and CpH8 have isolated.
06.11.2013 Tuesday
- • The isolation results are normal. CpH8s are goingto digest from EcoR1- Sp.
- • Long PCR have done for 8, EPIC and 500 Gibson A.
- • The Gibson primers have arrived for 8, EPIC and 6. diluted with TeBuffer.
- • 500 have diluted from the kit. The nucleic asid amount have measured on Nanodrop.
- • 6 EPIC and 9 EPIC's electrophoresis have done again. 6 EPIC is succeed but 9 EPIC failed. (01.00 am)
- • 1C3, EPIC and 8's PCR products' electrophoresis have done. 1C3 is true the others have not got band.
- • 8Z's transformation have done. (will be at 01.05 pm)
06.12.2013 Wednesday
- • 8 GC, 8 HF, EPIC GC, EPIC HF's PCR and electrophoresis have done. EPIC is succeed but 8 isn't. 8 needs long PCR.
- • 8, 8Z and RFP have transformated.(will be at 06.05 am)
- • 8 and EPIC's digestion and electrophoresis have done but no display.
- • We have waited arabinose but it didn't come.
- • No colonies from 8Z.
- • 7 Chl plates have prepared.
- • To learn is there any problem with primers because of there's no band on long PCR, the primer electrophoresis have done.
- I. The primers have walked on 250 V, 2.5 mins, %1 gel.
- II. EPIC F, 8R and 6R were pale. Wen could not figure out where is the problem, putting or diluting. We will ask tomorrow.
- • 8 has cut directly from BioBrick simple (5 µL) If the problem was on the band, it will dictly send and reported to iGEM.
- • The BioBrick simples have finished so we have diluted from AUC TURKEY team.
06.13.2013 Thursday
- • 8's long PCR and 6 gradient's PCR have done again.
- • 8's and 6's PCR's electrophoresis have done. 8 have failed again!!
- • Colonies have chosen from Loo and RLoo and their liquid cultures have done again.
- • We are going to continue with 64 fr
06.14.13 Friday
- • HO1, CHO1k, R8, RZ's PCR have done but unsuccesful.
- • With Omer Birgin's procedure.) HO1k, R8, 8 are transformated. (will be at 3.00.)
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Third Week of June
06.15.13 Saturday
- • 4, 5, 7, 9 have transformated. (will be at 05.30.)
- • 7, 9's PCR results' (16-17. 09.12 date) and 7, 9 iso results' dig electrophoresis have done.
- 7 old, 9 old (12.09.12)
- 7 iso, 9 iso
- 9(17), 9(16)
- 7 green, 7 purple
- • 9(17), 9(16), 7 green, 7 purple's electrophoresis have done again.
- • 4, 5, 7, 9 electrophoresis have done again.
- • 4, 5, 7, 9 PCR have done.PCR Pur done and digested.
06.16.13 Sunday
- • 4, 5, 7, 9 ligested. (9-4, 5-7)
- • 94, 57 transformated.
- • On the transformation from 5.30 4, 5 is unsuccesful.
- • 4, 5 transformated. (will be at 05.45 am)
- • 5, after PCR Pur cut simple's electrophoresis have done. (100 bp, 75 V, 20 min)
- • There is no colonies of 94,57 from 6.00pm.
- • 94, 57 transformated again. (will be at 11.10 am.)
- • 7, 9 liquid cultures have done19.50’de alındı.)
- • 7, 9 isolated.
- • pTetR PCR have done. Because of it has put into gel wrong the result is unsuccesful.
- • pTetR PCR Pur have done.
- • 7, 9 iso, pTetR PCR simples have digested.
06.17.13 Monday
- • 7, 9 iso, pTetR PCR dig simples' electrophoresis have done. 7, 9 succesful, pTetR unsuccesful(because of the gel.)
- • pTetR electrophoresis have done two times again.(75 V-25 min) unsuccesful.
- • pTetR have transformated. (will be at 8.40 pm.)
- • Because of there is no colonies of 4 and 5 from 05.45 am, it has transformed again. (10.25 pm)
- • 2012 pTetR iso simples have digested from ES. (75 V-25 min-10 bp) unsuccesful.
- • 94 have ligested with 500 and transformated. (will be at 03.55 am.)
- • Amp plates have prepared, two of them have given to AU
- • 57 PCR, 57 iso simples' liquid cultures have done. (will be at 1.45 am)
- • At 20.40, the pTetR plates have taken. Again there was empty bubles on it, just incase 3 liquidcultures have prepared.(will be at 11.00 am)
- • Because of there were empty bubles in most plates, for controle the transformation:
- i. From AUC, 2 NEB comp. have taken and a TOP 10 comp have taken from us.
- ii. 594 done from NEB10. RFP Amp done from a NEB 10 and a TOP 10.
- iii. There were not any Chl plates so we have borrowed from AUC, For 594 transformation plates are tomorrow will be at 2.55 pm.
- • There were still not colonies of 4 and 5 from 10.45 pm. There were empty bublles in it so we did not do a liquid culture.
18.06.13 Tuesday
- • 57 iso, PCR (1,2,3) simples have isolated.06.18.13 Tuesday
- • 94's liquid cultures have done.(will be at 10.45 pm)
- • 57 have digested, electrophoresis have done but results are unsuccesful.
- • From 3.00 pm 94 plates' liquid cultures have done.
- • 500, pTetR, pcons have digested.
- • 57, pTetR 7,pcons7 have connected with 500 and linner backbones.
- • From 10.45 pm isolation results' nanodrops have done.
- • The resultant 3 colonies of 94 plates' second one have digested.
- • CpH8 is trying again on antibiotic-free plates.(will be at 10.10 am)
06.19.13 Wednesday
- • 94 (2) have digested.
- • 57,pTetR7,pcons 7 have transformated. (will be at 5.35 pm)the liquid cultures have done. (will be at 8.20 am)
- • 94(2) electrophoresis have done, results are succesful.
- • 94's (second cultures) have isolated, digested and electrophoresis done. The results are succesful.
- • CpH8's liquid cultures have done. (will be at 3.23 am)
- • pLac LasI, LasI GFP PCR have done. Electrophoresis results are unsuccesful.
- • 13 Chl plates have prepared.
- • The liquid cultures from 3.23 am have isolated. Because of the nanodrop results were not good, just 8 1b hae digested. electrophoresis have done but results were unsuccesful.pLacI LasI and LasI GFP liquid cultures have not prepared because there were not any colonies in the plate.
- • LasI GFP have transformed again.(1 comp cell from NGSS, 1 comp cell from AUC)
- • 57 (500), pTetR7 liner, pCons7 liner have digested, the electrophoresis have done but results are wrong, 57(500) electrophoresis have done again but there is no band.
06.20.13 Thursday
- • 13 Chl plates have prepared.
- • The isolation of 03.23 am liquid cultures have done.
- • Because of the nanodrop results was not good, just CpH8 1b have digested but it is unsuccesful.
- • The liquid cultures of pLac, LasI, LasI(cfp) did not prepared because there were not any colonies.
- • LasI(cfp) have transformated again. (1 compt. Cell NGSS, 1 compt. Cell AUC)
- • 57(500), pTetR7(lineer), pcons7(lineer) have isolated. (The backbones were wrong except pTetR's.)
- • 57, 7 pcons7 have digested. 57 have tried again. the others are unsuccesful.
- • 57' electrophoresis have done again but there was not band.
- • 507 have ligested.
- • 507 have transformated. (will be at 8.00 am.)
- • Gfp, LasI liquid cultures have done. (will be at 2.45 pm.)
- • 594, 2, 2b, 1b's liquid cultures have prepared. (will be at 10.25 pm.)
- • Ecfp and LasI’s colonies have grew yesterday transformation's liquid cultures so it has put into the same broth with 594 1b. Later on although the controle, we realize the transformation did not happen.
06.21.13 Friday
- • The liquid cultures of 08.00 am 507 plates have done. (will be at 10.30 pm.)
- • LasI and CFP from 11.45 am have isolated and digested but the results are unsuccesful.
- • 507 have isolated.
-
Lab Notebook/June
First week of June
Notebook/First week
Second week of June
Notebook/Second week
Third week of June
Notebook/Third week
-
Colloboration
We had a meeting with AUC Turkey Team on 18th Of June at 2.00 pm. In this meeting teams explained their project mutually. After Presentations teams shared their positive and negative ideas about projects.
We had material exchange among two teams.
Cph8 was very important for our project, but although trying every way, we haven't got a positive result. So, we used cph8 in kits of AUC Turkey and Fatih Medical team
We received K769001 and 10407 genes from ATOMS2013 team; K772004 and K772005 genes from Fatih Medical team
While uploading our wiki to main page, we had some minor problems. So we got some help from AUC_turkey team.
Below you can find some photos
-
Fun!
One day of an iGEMer: "Experiment, wiki, experiment, wiki, experiment, wiki... zZzZzZz..."
Of course this is not true. We had lots of fun while we were studying.
Listening Ayşe Sister's guitar was a great pleasure for us. Also Dilara can play, but... In our free times, it is not valid actually, we played table tennis or took videos. The best parts of the days, definitely, meal times. Beril's mother bought us a baklava (turkish dessert, yummy!) for once :) Our traditional meal is pizza. Ordering pizza is cheaper than the others. We can choose "Secrets" as our favourite team song. Our favourite sport is wrestling, also.After three weeks that we stayed in the lab, we have collected some funny moments. Beyond them we took some videos else too. Enjoy it!"
-
Lab
Notebook
Here is the Notebook since January!
Procedures
If you want to learn how we did our experiments, you are in the right place
Safety
Safety have never been such funny!
-
Outreach
Adventures of E.coli and B.subtilis
Have you met E.Coli & B.Subtilis? ...
InfoStand at Mall
As a human practises work, we ...
bactUNO card game
As a human practises work, we ...
SynBio Day at the Kindergarden
We organized a SynBio day @kindergarden ...
Survey
As a human practises work, we ...
SynBio Presentation
As a human practises work, we ...
Introduction of SynBio to Companies
On SynBio projects, presentations ...
iGEM for Everyone
As a human practises work, we ...
NGSS Comics
As a human practises work, we ...
Colloborations
As a human practises work, we ...
Digestion
Materials:
- • Pcr Tube
- • Pipette tip which has filter
- • Plasmid which had isolated
- • -20 C EcoR1, Spel, Pstl, Xbal enzymes
- • ×10 NE Buffer 2
- • ×100 BSA
- • Nuclease Free H2O
- • Heat block or PCR
Experiment:
- • Take PCR tubes. Don’t forget to take tube for destination plasmid.
- • Paste the stickers to the tubes.
- • Lave NE Buffer 2, NF Water, BSA into ice when they melted.
- • Take the average of the nucleic acid concentrations measured by the spectrometer. Divide 500 by the DNA average.
- • Add 5µl Ne Buffer.
- • Add 0.5µl BSA Buffer.
- • Add 1 µl of the enzymes with barrier tips.
- • If you cut with Ecor1 and SpI, it will be up stream.
- • If you cut with Xbal and Pst1, it will be down stream.
- • Subtract the amount of DNA from 42.5 µl. This result will be the amount of NFW used.
- • Add the NFW with barrier tips and do one pippetting while taking the NFW.
- • Then the DNA is put into the PCR and is left there for 30 minutes.
ATTENTION!
- • Be sure enzymes are enough.
- • You must use plasmid which had isolated.
- • Use barrier tips has filter.
Plasmid Isolation
Materials:
- • Centrifuge
- • Liquid waste bin
- • 1,5 ml and 2 ml epps
- • Plasmid Isolation Kit
- • Resuspension Buffer
- • Lysis Buffer
- • Neutralization Buffer
- • Wash Buffer
- • Elution Buffer
- • Vortex
- • 50-500 µl pipettes
- • Timer
- • Filter columns and epps
- • PCR Tubes (mini epp)
Experiment:
- • Use gloves!
- • Liquid cultures centrifuge: 5 ml; 4100 rpm, 15 min. 1,5 ml; 13000 rpm, 10 min. (with 2 µl epp)
- • After the centrifuge, the supernatent should be disposed without taking any pellets along with it.
- • Add 250 µl Resuspension Solution. Vortex until to be homogeneous(about 30 sec.). You can pipetting too.
- • Add 250 µl Lysis Solution. You must invert(4-6 times).
- • There must be 3 min.
- • Add 350 µl Neutralization Solution and invert(4-6 times).
- • 5 min. centrifuge.
- • After centrifuge try to take supernatent without pellets as much as you can to fitler columns.
- • 1 min. (13 000 rpm) centrifuge. Throw the supernatent to liquid waste bin.
- • Add 500 µl Wash Solution. 1 min. centrifuge. Throw the supernatent to liquid waste bin.
- • Add 500 µl Wash Solution. 1 min. centrifuge. Throw the supernatent to liquid waste bin.
- • 1 min. centrifuge to get rid of alcohol.
- • Throw the supernatent with epp.
- • Put new 1,5 epp under the filter column.
- • Transfer the columns into 1.5 ml microcentrifuge tubes and leave their caps open for 2 minutes so that the ethanol that they contain dissolves in air.
- • Add 50 µl Elution Buffer.
- • Incubate for 2 minutes.
- • Centrifuge for 2 minutes.
- • Take the supernatent to new epp.
- • Measure them with Nanodrop.
Attention!
- • All centrifuges are 13000 rpm.
- • Don’t shake Lysis Solution.
LB-Agar
Materials:
- •LB-Agar(chemical storage)
- •Sterile dH2O
- •Flask
- •Liquid autoclave
- •Aluminium folio
- •Antibiotic
- •Empty plates
- •Parafilm
Experiment:
- •In a steril environment, the tare of the container should be measured and subtracted from the overall weight.
- •7 grams of LB Agar is put in the container.(If there is no LB agar, add the 4 gram LB broth and 2,7 gram agar.)
- •Add 200 ml distilled water to graduated cyclindar.
- •These two are mixed in a beaker.
- •When you are opening the beaker be sure that it doesn’t contact with air.
- •Autoclave tape is sticked on to the aliminium.
- •The beaker is placed in to the autoclave machine.
- •Autoclave machine has to have distilled water or demineralized water. The water level in the autoclave machine has to be a little higher than the liquid level of the beaker.
- •Then start the 90 min.
- •Take out the beaker and add antibiotics if required.
Attention!
- •Don’t be far away fire.
- •Don’ forget to write the date.
- •Don't forget add the amtibiotics
LB-Broth
Materials:
- •LB-Broth(chemical storage)
- •Sterile dH2O
- •Flask
- •Lab skale
- •Liquid autoclave
- •Alumininum folio
- •Antibiotic
Experiment:
- •In a steril environment, the tare of the container should be measured and subtracted from the overall weight.
- •4 grams of LB Broth is put in the container.
- •Add 200 ml distilled water to graduated cyclindar.
- •These two are mixed in a beaker.
- •When you are opening the beaker be sure that it doesn’t contact with air.
- •Autoclave tape is sticked on to the aliminium.
- •The beaker is placed in to the autoclave machine.
- •Autoclave machine has to have distilled water or demineralized water. The water level in the autoclave machine has to be a little higher than the liquid level of the beaker.
- •Then start the 90 min.
- •Take out the beaker and add antibiotics if required.
Attention!
- • Don’t be far away fire.
- • Don’ forget to write date
Ligation
Materials:
- • PCR Tube
- • Plasmids
- • 10× T4 DNA Ligase Buffer
- • T4 DNA Ligase
- • Nuklease Free H2O
- • Heat Block
Experiment:
- • Take PCR Tube ® Write Lig on it
- • Add 2µl upstream, 2µl downstream, 2µl destination plasmids
- • Add 2µl T4 DNA Ligase buffer
- • Add 1µl T4 DNA Ligase
- • Add 11µl nüklease free H2O. It must be 20 µl.
- • It must wait 37 C (10 min.), 80⁰C(20 min.)
- • After incubation you must followwith transformation. The only difference between normal transformation and lig’s transformation is adding 2µl plasmid.
Attention!
- • You must leave plasmid in -20°C after lig.
- • If electrophoresis results are successful you will follow with lig.
Liquid Culture
Materials:
- • Falkon
- • Pipette gun
- • 5-10 ml pipette
- • Ice box
- • LB-Broth
- • Antibiotic
- • Colony
- • Pipette
- • Shaker
- • Incubator
Experiment:
- In sterile environment,
- • Take falkons.
- • Add 5 ml LB-Broth into falkons with pipette gun.
- • Take an antibiotic into ice.
- • Add antibiotic.
- • Take the colony from plate.
- • Leave it into falkon.
- • Take falkons to the shaker.(it can be 240 or 320 rpm) Leave it 37⁰C ( 12-14 hours ) incubator.
Nano Drop
- • Open the program(Nano- Spech).
- • Choose dsDNA.
- • Be sure pipette is 2 µl.
- • Leave the drop middle.
- • First try Elution Buffer (for blanck)
- • Click blank.
- • If results are about 0(zero) its okay.
- • Write their names, click "start".
- • Nucleic Acid must be much over than 17 ng.
- You must check 2 times and to take their avarage.
Notes:
- Blanck = Elution Buffer
- Graph must be as straight as it can.
Transformation
Materials:
Notes:
- • 1.5 mL epps
- • Heat Block (42ºC)
- • Competent cells
- • LB-Broth
- • Ice
- • Ice Box
- • Microcentrifuge
- • Rocks for epp
- • Timer
- • LB agar plates
- • Spreader
- • Shaker (240 rpm)
- • Parafilm
- • Band
- • 1 µl pipette (for plasmid)
- • 50µl pipette
- • 150 µl pipette (for spread)
- • 450 and 500 µl pipettes (for LB)
- • Incubator (37 C)
- • Alcohol
- • If there is diluting from the kit:
- • Nuclease Free H2O
- • 15 µl pipette
- • PCR Tube (Mini epp)
Experiment:
Notes:
- • Sterilize the environment.
- • Take the competent cells from -80°C.
- • Turn on the heat block (42⁰C)
- • Put 1 µl plasmid in a 1,5 ml ep.
- • Put competent cells in the same 1,5 ml epp. Use 50µl competent cells.
- • Place the epps into the microcentrifuge. (30-60 sec., 3000 rpm)
- • Incubate on ice for 45 min.
- • Put epps into the heat block at 42⁰C for 120 sec.
- • Put epps back on ice for 5 min. to reduce damage to the e.coli cells.
- • Add 450 ul of LB(with no antibiotic added)
- • Incubate examples on shaker for 1 hour at 37⁰C. (240 rpm)
- • Spread about 135 ul of the final examples on LB plates (with appropriate antibiotic added-usually Ampicillin or Kanamycin)
- • Incubate overnight.
- • Pick colonies about 14-16 hours later
ATTENTION
Notes:
- • Don’t use gloves when you are working with fire.
- • Don’t forget to write notes on epps.
- • Be quick while sirkini Wilhelm compatent cells
Attributions
Attributions
All of us work hard for iGEM 2013. But our particular thanks go to the esteemed people and foundations behind the scenes...
Firstly we want to thank our school, Ankara Private Atlantik Nevin Gökçek Science High School , to give us such opportunities.
Our very special thanks go to...
Wet Lab Works
...Turgut Ozal University (former Fatih) for providing their labs and other facilities for us. We also want to thank our head advisor Assoc. Prof. Esra Gündüz who was ready at anytime to support us.
...Sentegen for helping us to to provide our genes, special thanks for cool centrifuge.
...the whole team, especially to our advisors: Fatma Betül Çevik, Ayşe Demirci, Esma Ölmez who were always with us.
Also we want to thank to Muradiye Acar, Burak Yılmaz, Fazilet Yılmaz, Gökhan Nas, Ömer Faruk Hatipoğlu for their helps about our Project subject also our experiments. And thanks to genetic students for their helps about our experiments.
Wiki
... Havva Gulay Gurbuz – graduate student at Bilkent University. She spent a lot of her time for us.
... Busra Nur Aydın who helped us with designing - she has magical hands.:)
.. Kasım Özkan who helped us with doing our comics.
Funding
We want to thank Osman Birgin , who is the head of Energy Market Regulatory Authory for finding us generous funds. He did things which mean a lot for us.
We want to thank our school again in this chapter. They provided their sources for us.
Beyond these, we want to thank Turgut Ozal 2013 Collegiate iGEMers for being our advisors. We also want to give a special thank to Esin and Kübra Sisters for yummy meals :)
There is also one more group that we want to thank. They were supporting us in our desperative times. Our parents! Especially to Beril’s mom Hülya Gürdap for baklava (Turkish dessert) and to Dilara’s mom Özlem Soylu for transportation.
Aaaand finally: Our instructor Mihriban Ozgun for her amazing "instructorhood"
Team:NGSS AEI TURKEY
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- | <div class="picHold"><a | + | <div class="picHold"><a rel="Appendix" href="#!/pageFun"><span class="zoomSp"></span><img src="https://static.igem.org/mediawiki/2013hs/a/a4/I9.jpg" alt=""></a></div> |
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+ | <div class="box"> | ||
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+ | <div class="containerContent"> | ||
+ | <h2>Team Video</h2> | ||
+ | <div class="col1"> | ||
+ | <div class="Btns1"> | ||
+ | <a href="#" class="upBtn"></a> | ||
+ | <a href="#" class="downBtn"></a> | ||
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+ | <object width="560" height="315"><param name="movie" value="https://www.youtube-nocookie.com/v/CiI7UamwMUI?hl=tr_TR&version=3&rel=0"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="https://www.youtube-nocookie.com/v/CiI7UamwMUI?hl=tr_TR&version=3&rel=0" type="application/x-shockwave-flash" width="560" height="315" allowscriptaccess="always" allowfullscreen="true"></embed></object> | ||
+ | </div> | ||
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<object width="560" height="315"><param name="movie" value="https://www.youtube-nocookie.com/v/CiI7UamwMUI?hl=tr_TR&version=3&rel=0"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="https://www.youtube-nocookie.com/v/CiI7UamwMUI?hl=tr_TR&version=3&rel=0" type="application/x-shockwave-flash" width="560" height="315" allowscriptaccess="always" allowfullscreen="true"></embed></object> | <object width="560" height="315"><param name="movie" value="https://www.youtube-nocookie.com/v/CiI7UamwMUI?hl=tr_TR&version=3&rel=0"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="https://www.youtube-nocookie.com/v/CiI7UamwMUI?hl=tr_TR&version=3&rel=0" type="application/x-shockwave-flash" width="560" height="315" allowscriptaccess="always" allowfullscreen="true"></embed></object> | ||
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+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li id="pageModule1Abstract"> | ||
+ | <div class="box"> | ||
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+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | <div class="containerContent"> | ||
+ | <h2 style="font-size:32px">Photo Communication - Abstract</h2> | ||
+ | <div class="col1"> | ||
+ | <div class="Btns1"> | ||
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+ | |||
+ | <p class="padBot4">Communication between bacteria is a very commonly used phenomenon . It is a system which is continuously tried to enhanced and developed. As an alternative to these mostly AHL/inducible promoter dependent communicaiton systems we designed a pathway that targets messaging between different kinds of bacteria and especially bacteria in different and independent media. In our project we are going to examine the ability of a bacterium to affect other colonies via light/color production and emission as a product of several biochemical activities and additionally the effectiveness for different colors and wavelenghts.</p> | ||
+ | <p class="padBot4">Furthermore, for the safety part of the project, we will analyze the capability of colonies to induce the death of other colonies or activating a second antibiotic resistance via ‘’photocommunication’’ additionally to the already integrated one in the backbone against contamination risks.</p> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li id="pageModule2Abstract"> | ||
+ | <div class="box"> | ||
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+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | <div class="containerContent"> | ||
+ | <h2 style="font-size:32px">Murder Communication - Abstract</h2> | ||
+ | <div class="col1"> | ||
+ | <div class="Btns1"> | ||
+ | <a href="#" class="upBtn"></a> | ||
+ | <a href="#" class="downBtn"></a> | ||
+ | </div> | ||
+ | <div class="scroll1"> | ||
+ | |||
+ | <p class="padBot4">As a result of rising number of studies in the field of SynBio the role of genetically modified organisms in our lives increased and gained an important role. At the same time, to keep control of the interaction we get with them and to take precautions against possible dangers, various safety modules were developed. We also established safety system with the help of quorum sensing in our project which is focused on bacterial interactions. At this point of idea, we used LasI and LasR; the communication chemicals of Pseudomonas Aeruginosa. Response of the receiver cell will be the output of holin-endolysin enzymes to the interaction with LasI gene which will be produced by sender cell. These enzymes will cause the perforation of bacterium cell membrane system and this will result with bacterium death. Consequently, a controlled cell death pathway will be developed with the system of an inducer connected to the sender.</p> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | |||
+ | <li id="pageProjectAbstract"> | ||
+ | <div class="box"> | ||
+ | |||
+ | <div class="closePlane"> | ||
+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | |||
+ | <div class="containerContent"> | ||
+ | <h2>Abstract</h2> | ||
+ | <div class="col1 padBot3"> | ||
+ | <div class="col4 magRight2"> | ||
+ | <h3><a class="_link3" href="#!/pageModule1Abstract">Module 1 - PhotoCommunication Abstract</a></h3> | ||
+ | <p>Communication between bacteria is a very commonly used ... <a class="_link3" href="#!/pageModule1Abstract">Read More</a></p> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | <div class="col1"> | ||
+ | <div class="col4 magRight2"> | ||
+ | <h3><a class="_link3" href="#!/pageModule2Abstract">Module 2 - MurderCommunication Abstract</a></h3> | ||
+ | <p>As a result of rising number of studies in the field of SynBio ... <a class="_link3" href="#!/pageModule2Abstract">Read More</a></p> | ||
+ | |||
+ | |||
+ | </div> | ||
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+ | </div> | ||
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+ | </div> | ||
+ | <div class="containerContent"> | ||
+ | <h2>Notebook</h2> | ||
+ | <div class="col1"> | ||
+ | <div class="Btns1"> | ||
+ | <a href="#" class="upBtn"></a> | ||
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+ | <iframe src="https://www.google.com/calendar/embed?height=500&wkst=1&bgcolor=%23FFFFFF&src=ngss.aei.turkey%40gmail.com&color=%232952A3&ctz=Europe%2FIstanbul" style=" border-width:0 " width="500" height="500" frameborder="0" scrolling="no"></iframe> | ||
+ | </div> | ||
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+ | </div> | ||
+ | |||
+ | <div class="containerContent"> | ||
+ | <h2>Results</h2> | ||
+ | <div class="col1 padBot3"> | ||
+ | <div class="col4 magRight2"> | ||
+ | <h3><a class="_link3" href="#!/pageModule1Result">Module 1 - PhotoCommunication Abstract</a></h3> | ||
+ | <p>PhotoCommunication - Result <a class="_link3" href="#!/pageModule1Result">Read More</a></p> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | <div class="col1"> | ||
+ | <div class="col4 magRight2"> | ||
+ | <h3><a class="_link3" href="#!/pageModule2Result">Module 2 - MurderCommunication Abstract</a></h3> | ||
+ | <p>MurderCommunication - Result <a class="_link3" href="#!/pageModule2Result">Read More</a></p> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | |||
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+ | </div> | ||
+ | <div class="containerContent"> | ||
+ | <h2 style="font-size:32px">PhotoCommunication - Result</h2> | ||
+ | <div class="col1"> | ||
+ | <div class="Btns1"> | ||
+ | <a href="#" class="upBtn"></a> | ||
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+ | |||
+ | <p class="padBot4"> We have started with cloning the both genes for our communication system which depends on Luciferase and Cph8. We went on two ways to do this. We did the PCRs of the primers which were designed for the genes in iGEM distribution kit. In the meantime, we did the transformation of them into E.Coli TOP10 strain.</p> | ||
+ | <p class="padBot4">We completed the cloning of the Luciferase system by using the parts BBa_K325219 (Red Luc.), BBa_K325108 (EPIC Firefly Luc.) and BBa_K325909 (Lux Operon) submitted by the Cambridge Team 2010; to make the sender system. For the continuation of experiments, we waited for CpH8 results.</p> | ||
+ | <p class="padBot4">To establish the response system which is related to the CpH8 induced pOmpC promoter in the receiver system of PhotoCommunication,; we decided to use two different assembly methods for Luciferase(BBa_K325108) and GFP(BBa_K769001). These were Gibson and 3A Assembly methods. The system we aimed to build was Cph8+pOmpC+Luciferase/GFP. Regarding the fact that we have to combine this triple with the pSCB1C3 backbone after their successful ligation, we decided to continue with Gibson Assembly. We didn’t have experience before with using the Gibson Assembly method thus, we designed primers just for one system and ordered it. We were thinking of using Gibson Assembly on the other systems according to the results of the first trials. The Gibson Assembly master mix kit was sent before by iGEM high school teams sponsor NEB.</p> | ||
+ | <p class="padBot4">Before we had Gibson Assembly primers, we tried to multiply Cph8, pOmpC, EPIC Luc. and GFP device systems via transformation and PCR procedures. We were thinking to do the 3A Assembly in this way. We obtained all genes with PCR except for Cph8 and EPIC Luc. because of their extended length (Figure 1). We continued with pOmpC, GFP Device and pLac attained from PCR samples. At the end of the transformation oof these parts, isolation digestion followed in order to obtain EPIC Luc. and we passed on to the 3A assembly of EPIC Luc. and pOmpC.</p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/1/1b/Imagea1.png" width="500px" alt=""></div> | ||
+ | <p class="padBot4">pLacI(9): 220 bp, Cph8(8): 4333 bp, EPIC: 2626 bp, pOmpC(6): 108 bp, pOmpC’li GFP Device(3): 990 bp.9, PCRs of 3 and 6 are done</p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/b/b5/Imagea2.png" width="500px" alt=""></div> | ||
+ | <p class="padBot4">BBa_K769001: 990 bp. We continue with the 3 and 3(2).</p> | ||
+ | <p class="padBot4">For 3A Assembly we needed first to ligate pOmpC and EPIC Luciferase. For this, we did the ligation to pSB1C3 from the sections of isolation and PCR purification samples . In the meantime, we tried to combine EPIC Luc. with pLac to show its working. Likewise for that combination we used isolation and PCR samples. To sum up, there were successful results for the ligation of the pOmpC promoter and EPIC Luc. which we called ‘’6’’, but the ligation of pLac promoter and EPIC Luc. again which we called this time ‘’9’’, can hardly be regarded as a success. </p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/a/ad/Imagea3.png" width="500px" alt=""></div> | ||
+ | <p class="padBot4">pOmpC(6): 108 bp, EPIC Luc: 2626 bp, pLac(9): 220 bp. pOmpC combined with EPIC Luciferase</p> | ||
+ | <p class="padBot4">After pOmpC and EPIC Luc. were combined, the GFP system with pOmpC was restricted appropriately and became ready to ligation, we focused on the CpH8’s cloning to add it to the top of the system.</p> | ||
+ | |||
+ | <p class="padBot4">We tried transformation again after decreasing the antibiotic concentration in the plates. There were few colonies in some plates while some of them didn’t have any colonies. We grew those colonies in decreased antibiotic liquid cultures. Unfortunately, the digestion results did not give the amount we wanted and we thought they could be the product of contamination of colonies. </p> | ||
+ | |||
+ | <p class="padBot4">We repeated transformation by taking NEB10 compotent cells which were confirmed by AUC_Turkey before, to see if the problem is about our compotent cells . The result did not change and there still weren’t any colonies.</p> | ||
+ | |||
+ | <p class="padBot4">By the way, our Gibson Assembly primers have arrived. We thought we could reproduce Cph8 in that way so we have started PCR procedure. Primarily, we did PCR on Cph8 and EPIC Luc. with long PCR enzyme system because of their extended length. We could not get any results from both of these elements (Figure 4).We did PCR for EPIC Luc. and CpH8’s with High Fidelity, too. This time, we were able to reproduce EPIC Luc. (Figure 5). In the meantime, we were doing PCR of CpH8 from iGEM BioBrick kit because we were not able to reproduce it before. Our kit was not enough to do such numbers of PCRs and transformations so we diluted this plasmid from 2013 AUC_Turkey and 2012 Fatih Medical teams kits next to ours. We did the long PCR of CpH8 with Golden Blocked PCR hoping for efficiency increase (Figure 6). Again there was no band on the electrophoresis gel after PCR.</p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/4/44/Imagea4.png" width="500px" alt=""></div> | ||
+ | <p class="padBot4">1C3: pSB1C3(2070 bp) </p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/0/03/Imagea5.png" width="500px" alt=""></div> | ||
+ | <p class="padBot4">In this PCR, although EPIC was reproduced , Cph8 couldn't obtained</p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/e/ec/Imagea6png.png" width="500px" alt=""></div> | ||
+ | <p class="padBot4">Result of Cph8 Long PCR in golden block and Result of pOmpC Gibson Primerlers Gradient PCR </p> | ||
+ | <p class="padBot4">We did electrophoresis of restricted and unrestricted CpH8 on % 0.8 gel and 75V for 1 h to control the presence of gene in the matter we diluted. Again we did not see any band on the electrophoresis gel </p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/d/de/Imagea62.png" width="500px" alt=""></div> | ||
+ | <p class="padBot4">M30109 Cph8 sistemi çoğaltılamayınca parçalardan bütüne gitme amacıyla HO1&PcyA, r8 pcr yapıldı. Aynı zamanda kitte bulunan Cph8'in LacZ'li halinin(8z) de pcr ı yapıldı.8z->BBa_K322327: 4027 bp, r8->BBa_K322124: 2253 bp, HO1->BBa_K098010: 1531 bp. </p> | ||
+ | <p class="padBot4">We have optimized our transformation protocol after consult to Turgut Ozal University Medical School Medical Genetics Department professors and transformed CpH8 again with a new protocol, but we still could not get any colonies. Although our 14 day and night reproduce working, we could not get any results for CpH8 so we gave up trying this system and started to wait for the delivery of our designed CpH8 device which includes Ampicillin resistance. </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li id="pageModule2Result"> | ||
+ | <div class="box"> | ||
+ | <div class="closePlane"> | ||
+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | <div class="containerContent"> | ||
+ | <h2 style="font-size:32px">MurderCommunication - Result</h2> | ||
+ | <div class="col1"> | ||
+ | <div class="Btns1"> | ||
+ | <a href="#" class="upBtn"></a> | ||
+ | <a href="#" class="downBtn"></a> | ||
+ | </div> | ||
+ | <div class="scroll1"> | ||
+ | |||
+ | <p class="padBot4">In our mass killing system which depends on the quorum sensing, firstly, we started with controlling the genes we got from Team Fatih Medical 2012. We did the measurement and digestion of the isolation samples of Fatih Medical. We also did digestion of the PCR purification samples and controlled them </p> | ||
+ | |||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/e/ea/15.06.13_kesik.png" width="500px" alt=""></div> | ||
+ | <p class="padBot4">BBa_K772005, 8:BBa_K772004</p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/2/2e/17.06.13_dig%28iso%29_kesik.png" width="500px" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/7/71/15.06.13_PCR_kesik.png " width="500px" alt=""></div> | ||
+ | <p class="padBot4">We thought to build an IPTG controlled sender system by adding a constitutive promoter at the downstream of the LasI system. We did PCR of BBa_J23100 and transformation for the same part, alternatively we tried pTetR (BBa_R0040) and BBa_J23101, too. We tried to combine these promoter alternatives and our LasI system with 3A Assembly but we could not get successful result. Whereupon we decided to reproduce and try BBa_K084007 pLac bound LasI system of Team Chiba 2008 which was added to the distribution kit and BBa_I0407 LasI series which includes ecfp under pTetR. In the meantime, we controlled the functionality of LasR-T4 Lysis system by using the LasI series (K772005) of Team Fatih Medical 2012 without adding promoter to the downstream region of LacI and by using it as a device producing LasI under the control of pLac.</p> | ||
+ | <p class="padBot4">To sum up, with a chemical (IPTG) inducible quorum sensing, it is possible to kill whole receiver cells in desired time. </p> | ||
+ | <p class="padBot4">In the meantime BBa_J23100 (5) and BBa_K772005 (7) are ligated successfully (57). LasR device BBa_K772004 (9) and T4 Lysis Device BBa_K112808 are also ligated with success.</p> | ||
+ | <p class="padBot4">As a result, we completed our composite parts; confirmation experiments are continuing.</p> | ||
+ | |||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/c/c9/15.06.13_pcr_pro_kesik.png" width="500px" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/b/bf/19.06.13_dig_94_%281-2-3%29_kesik.png" width="500px" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/a/a9/19.06.13_dig_94_kesik.png" width="500px" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/7/77/20.06.13_dig_57_kesik.png" width="500px" alt=""></div> | ||
+ | <p class="padBot4">Ligation Results of K990002 and K990005 </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li id="pageModule1Assembly"> | ||
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+ | </div> | ||
+ | <div class="containerContent"> | ||
+ | <h2 style="font-size:32px">PhotoCommunication - Assembly/Design</h2> | ||
+ | <div class="col1"> | ||
+ | <div class="Btns1"> | ||
+ | <a href="#" class="upBtn"></a> | ||
+ | <a href="#" class="downBtn"></a> | ||
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+ | <div class="scroll1"> | ||
+ | |||
+ | <p class="padBot4">For the installation of an interaction system which is independent from any chemicals or the medium, we used the photoreceptor called Cph8 in the literature[3]. This receptor was first used by Team UT Austin in 2004. In order to harvest the molecule PCB which is one of the most important members of the Cph8 pathway, we added to our final part the HO1 (heme oxygenase) and PcyA (phycocyanobilin=ferredoxin oxireductase) sequences which are responsible for the synthesis of PCB. As PCB molecule gets in contact with light and induces the Cph8-complex, the inner cell element of the complex, EnvZ, activates the OmpR protein by phosphorylation. The phosphorylated OmpR induces hereupon the pOmpC promoter and the genes in the upstream region can be translated.</p> | ||
+ | <p class="padBot4">3. ^ Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, Davidson EA, Scouras A, Ellington AD, Marcotte EM, Voigt CA (2005). "Synthetic biology: engineering Escherichia coli to see light".Nature 438 (7067): 441–2. doi:10.1038/nature04405. PMID 16306980.</p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/b/bd/Assembly.png" width="500px" alt=""></div> | ||
+ | <p class="padBot4">In this way, we can use extracellular light to regulate intracellular events. In order to test the intercellular applicability of this regulable procedure, we built system (A). System (B) was built to analyze the practicability of the resistance gain in the safety field and other fields besides the light controllable backbone during the life cycle of bacteria. </p> | ||
+ | <p class="padBot4">System A: </p> | ||
+ | <p class="padBot4">As a sender system we need a colony which is capable of producing a light stimulus as the basic element of the communication. Because the Cph8 system works best at 660 nm(red light) we transformed our sender cells witht he red luciferase part(BBa_K325219) designed by Team Cambridge 2010.</p> | ||
+ | <p class="padBot4">To get concrete results from the receiver cell which will respond to the photostimulus, it was a safe way to use proteins which are simply visible through their color. In the upstream region of the OmpR inducible pOmpC promoter we placed:</p> | ||
+ | <p class="padBot4">a. EPIC Luciferase(BBa_K325100) ya da </p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/2013hs/d/d4/Light_Responsive_System.png" width="500px" alt=""></div> | ||
+ | <p class="padBot4">b. GFP(BBa_K769001)</p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/2013hs/4/4e/Right_Regulator_System_pOmpC_GFP.png" width="500px" alt=""></div> | ||
+ | <p class="padBot4">and intended to test the GFP or EPIC Luciferase respond of the receiver bacteria in case of a Red Luciferase stimulus from the sender bacteria.</p> | ||
+ | <p class="padBot4">System (B):</p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/2013hs/0/02/2879_bp.png" width="500px" alt=""></div> | ||
+ | <p class="padBot4">Here, our goal was to find out whether it is possible to strengthen the bacterial antibiotic resistance in the presence of light or - another possibility – whether we could terminate the bacterial activity of the receiver at a desired time by disabling the light source.</p> | ||
+ | <p class="padBot4">This was established by setting the second antibiotic resistance gene upstream of the pOmpC promoter in the receiver cell containing Cph8. In this way, if we expose the receiver to light (~660 nm) it becomes resistant to Ampicillin in our case (in addition to the Chl resistance contained in pSB1C3 plasmid backbone) and the risk of contamination by undesired colonies will be eliminated. On the other hand, if we stop the exposure our modified bacteria won’t be resistant anymore to Ampicillin and will go down with the other ones because of the still remaining Ampicillin in the medium.</p> | ||
+ | <p class="padBot4">Our Parts</p> | ||
+ | <table border="4"> | ||
+ | <tr> | ||
+ | <td><p class="padBot4">pOmpC+Epıc</p></td> | ||
+ | <td><a href="http://parts.igem.org/Part:BBa_K990001" target="_blank"><p class="padBot4">BBa_K990001</p></a></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><p class="padBot4">HO1+PcyA+pOmpC+Amp Res.</td> | ||
+ | <td><a href="http://parts.igem.org/Part:BBa_K990003" target="_blank"><p class="padBot4">BBa_K990003</p></a></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><p class="padBot4">Cph8 System</td> | ||
+ | <td><a href="http://parts.igem.org/Part:BBa_K990004" target="_blank"><p class="padBot4">BBa_K990004</p></a></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li id="pageData"> | ||
+ | <div class="box"> | ||
+ | |||
+ | <div class="closePlane"> | ||
+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | |||
+ | <div class="containerContent"> | ||
+ | <h2>Data Page</h2> | ||
+ | <div class="col1 padBot3"> | ||
+ | <div class="col4 magRight2"> | ||
+ | <h3><a class="_link3" href="#!/pageModule1Data">Module 1 - PhotoCommunication Data</a></h3> | ||
+ | <p>Data Page for PhotoCommunication ... <a class="_link3" href="#!/pageModule1Data">Read More</a></p> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | <div class="col1"> | ||
+ | <div class="col4 magRight2"> | ||
+ | <h3><a class="_link3" href="#!/pageModule2Data">Module 2 - MurderCommunication Data</a></h3> | ||
+ | <p>Data Page for MurderCommunication ... <a class="_link3" href="#!/pageModule2Data">Read More</a></p> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li id="pageModule1Data"> | ||
+ | <div class="box"> | ||
+ | <div class="closePlane"> | ||
+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | <div class="containerContent"> | ||
+ | <h2 style="font-size:32px">Module1 : PhotoCommunication - Data Page</h2> | ||
+ | <div class="col1"> | ||
+ | <div class="Btns1"> | ||
+ | <a href="#" class="upBtn"></a> | ||
+ | <a href="#" class="downBtn"></a> | ||
+ | </div> | ||
+ | <div class="scroll1"> | ||
+ | |||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/2013hs/5/50/1Modul1.PNG" width="500px" alt=""></div></td> | ||
+ | <p class="padBot4">607 nm light expands from sender cell with the help of red luciferase gene.</p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/2013hs/e/eb/1Modul2.PNG" width="500px" alt=""></div></td> | ||
+ | <p class="padBot4">Through the Cph8 system, haem oxygenase and phycocyanobilin: ferredoxin oxidoreductase enzymes get activated and PCB synthesis begins. Light activated PCB induces Cph1 and histidin kinase effect of EnvZ gets activated. OmpR gets phosphorylated with the effect of Kinase. Phosphorylated OmpR alerts and activates pOmpC. With the operation of Cph( system, as a response to red luciferas stimulant:) the response is recieved at bright light with EPIC luciferase</p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/6/6f/1Modul3.PNG" width="500px" alt=""></div></td> | ||
+ | <p class="padBot4">the response is recieved with GFP</p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/4/4c/1Modul4.PNG" width="500px" alt=""></div></td> | ||
+ | <p class="padBot4">Amp resistance also gets activated</p> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li id="pageModule2Data"> | ||
+ | <div class="box"> | ||
+ | <div class="closePlane"> | ||
+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | <div class="containerContent"> | ||
+ | <h2 style="font-size:32px">Module2 : MurderCommunication - Data Page</h2> | ||
+ | <div class="col1"> | ||
+ | <div class="Btns1"> | ||
+ | <a href="#" class="upBtn"></a> | ||
+ | <a href="#" class="downBtn"></a> | ||
+ | </div> | ||
+ | <div class="scroll1"> | ||
+ | |||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/2013hs/5/5b/2Module1.PNG" width="500px" alt=""></div></td> | ||
+ | <p class="padBot4">Under constitutive promoter, LacI synthesis represses pLac working.</p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/2013hs/2/2b/2Modul2.PNG" width="500px" alt=""></div></td> | ||
+ | <p class="padBot4">LacI influence represses by adding IPTG to the medium and in our sender cells LasI produce starts and spreads.</p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/2013hs/c/c5/2Modul3.PNG" width="500px" alt=""></div></td> | ||
+ | <p class="padBot4"> In receiver cells, under control of constitutive promoter, lasR is produced constantly</p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/2013hs/2/22/2Modul4.PNG" width="500px" alt=""></div></td> | ||
+ | <p class="padBot4">The LasI's connects with LasR molecules in receiver cells and activates pLas. </p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/2013hs/c/c8/2Modul5.PNG" width="500px" alt=""></div></td> | ||
+ | <p class="padBot4">Holin and endolysin reproduces by reading T4 lysis device.</p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/2013hs/5/5c/2Modul6.PNG" width="500px" alt=""></div></td> | ||
+ | <p class="padBot4">Endolysin drills from an outside layer so the bacterium begins to die. </p> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li id="pageAtAGlance"> | ||
+ | <div class="box"> | ||
+ | <div class="closePlane"> | ||
+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | <div class="containerContent"> | ||
+ | <h2>At a Glance</h2> | ||
+ | <div class="col1"> | ||
+ | <div class="Btns1"> | ||
+ | <a href="#" class="upBtn"></a> | ||
+ | <a href="#" class="downBtn"></a> | ||
+ | </div> | ||
+ | <div class="scroll1"> | ||
+ | <p class="padBot4"> Photo Communication - At A Glance </p> | ||
+ | <p class="padBot4">Our project consists mainly of two modules. These modules basically depend on the communication between bacteria. Our main target was communication via light without the need of chemicals as intermediate agents. Besides this, we have also built a collective killing system which is controllable with chemical inducers.</p> | ||
+ | |||
+ | <p class="padBot4">In our PhotoCommunication module, we used the Luciferase system, the bioluminescence genes from fireflies which were added to the partsregistry in 2010 by Team Cambridge. Regarding the fact that our receiver device works best at around 660 nm wavelength which represents red light, we installed Red Luciferase into our sender cells (BBa_K325219).</p> | ||
+ | |||
+ | |||
+ | <p class="padBot4">In the head member of the PhotoCommunication module – the receiver bacteria – we applied the Cph8 photosensitive device (BBa_M30109) as photoreceptor which was submitted in the partsregistry by Team UT_Austin in 2004. In this system, membrane proteins PCB and Cph1 interact in the case of light exposure. As a result the histidine kinase enzyme activity of another membrane protein EnvZ gets activated and phosphorylates the endogenous transcription factor OmpR. Hereupon, OmpR induces pOmpC promoter and right after light/Cph8 activation the promoter activity of pompC starts.</p> | ||
+ | |||
+ | |||
+ | <p class="padBot4">We built several systems in the PhotoCommunication module for different feedbacks of the receiver realted to the stimulus:</p> | ||
+ | |||
+ | |||
+ | <p class="padBot4">1) The Luciferase Respond: The receiver induced by the red light from the sender, answers with the blue light which is produced by the Luciferase translated under the activated pOmpC promoter. This will be the visual proof of bacterial communication.</p> | ||
+ | |||
+ | <p class="padBot4">2) The GFP Respond: This time, the receiver answers by producing green fluorescent protein (GFP) instead of the Luciferase system.</p> | ||
+ | |||
+ | |||
+ | <p class="padBot4">3) Control of Antibiotic Resistance: The purpose here is that colonies show a second antibiotic resistance in a bright environment (appropriate wavelength is required) in addition to that one which is already integrated in the backbone due to the sequence coding for Ampicillin-resistance under the pOmpC promoter. On the contrary, the same colonies shall lose their extra antibiotic resistance in case of interruption of the light stimulus. </p> | ||
+ | |||
+ | <p class="padBot4"> Murder Communication - At A Glance </p> | ||
+ | <p class="padBot4">In module 2, we did a communication system which depends on quorum sensing. For this, we used LasI-LasR which is a communication chemical of the gram(-) bacteria called P. Aeruginos. This system works with a similar to interaction between LuxI and LuxR mechanism. The LasI produced by sender gets out of the cell and connects with LasR in receiver cells.The LasI-R complex activates pLas promoter and the sender controlled pathway in receiver cells starts working. Thus, we decided to use this system to kill the colonies which switch on their reciever mode at any time we want. We added T4 lysis device after pLas in reciever cells. T4 lysis device; provides the puncture of the cell from inside with holin enzyme and from outside layer with the endolysin enzyme. Thus, LasI coming from sender activates the pathway that will kill the receiver cell by ensuring the rupture of the cell membrane of the receiver bacteria.</p> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li id="pageModule2Assembly"> | ||
+ | <div class="box"> | ||
+ | <div class="closePlane"> | ||
+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | <div class="containerContent"> | ||
+ | <h2 style="font-size:32px">Murder Communication - Assembly/Design</h2> | ||
+ | <div class="col1"> | ||
+ | <div class="Btns1"> | ||
+ | <a href="#" class="upBtn"></a> | ||
+ | <a href="#" class="downBtn"></a> | ||
+ | </div> | ||
+ | <div class="scroll1"> | ||
+ | |||
+ | <p class="padBot4">In view of the enormous problems we had with the cloning of Cph8 parts and the fact that our self-designed Cph8 based genes will arrive much later than expected, we decided to develop an alternative project to PhotoCommunication. We started with thinking whether there was a way to make a new project based on our knowledge from the research on bacterial communication methods. As a result of our brain stormings, we came to the result that we want to establish a killer system and link it to the quorum sensing pathway in our former project ideas.</p> | ||
+ | <p class="padBot4">There was the option for us to use the optimized quorum sensing LasI/R genes of Team Fatih Medical 2012. Finally, we used the LasI/R quorum sensing mechanism of the gram(-) P.Aeruginosa and combined IPTG-inducible LasI production in the sender bacteria with the LasI/LasR dependent T4 Lysis device in the receiver bacteria in order to induce the death of the receiver.</p> | ||
+ | <p class="padBot4">We used here for the sender BBa_K772005 from Team Fatih Medical 2012. In order to be able to control this device with LacI and IPTG, we decided to add a constitutive promoter at the downstream region of LacI. For this purpose, we ligated part BBa_K772005 separately with these promoters: BBa_J23100, pTetR and BBa_J23101. In this way, we got a sender which produces LacI constitutively and inhibits the transcription of LasI at the upstream region of by repression of pLac. After adding IPTG, the inhibitory effect of LacI on pLac will disappear and the production of LasI can start.</p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/2013hs/9/93/9_K772004.png" width="500px" alt=""></div></td> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/2013hs/1/12/7_K772005.png" width="500px" alt=""></div></td> | ||
+ | |||
+ | <p class="padBot4">For the receiver we also used a part from Team Fatih Medical 2012: BBa_K772004.Las Receiver Device. This time, the constitutive promoter pTetR is at the downstream of the LasR gene and ensures the constant production of LasR so that it can interact any time if a LasI molecule from the sender arrives. After interaction, LasI/LasR complex activates pLas promoter and thereby the translation of the upstream genes. At this point, we chose T4 Lysis Device which will be responsible for the bacterial cell death and worked with BBa_K112808 designed by Team UC Berkeley 2008. In conclusion, we have built a quorum sensing inducible lysis system by joining these two parts.</p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/2013hs/0/02/T4_Lysis_Device_K112805.png" width="500px" alt=""></div></td> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/f/ff/Birle%C5%9Fik.png" width="500px" alt=""></div></td> | ||
+ | <p class="padBot4">Our Parts</p> | ||
+ | <table border="4 "> | ||
+ | <tr> | ||
+ | <td><p class="padBot4">IPTG Inducible LasI Device</p></td> | ||
+ | <td><a href="http://parts.igem.org/Part:BBa_K990002" target="_blank"><p class="padBot4">BBa_K990002</p></a></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><p class="padBot4">LasR Device+T4 Lysis</p></td> | ||
+ | <td><a href="http://parts.igem.org/Part:BBa_K990005" target="_blank"><p class="padBot4">BBa_K990005</p></a></td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li id="pageProjectAssembly"> | ||
+ | <div class="box"> | ||
+ | |||
+ | <div class="closePlane"> | ||
+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | |||
+ | <div class="containerContent"> | ||
+ | <h2>Assembly/Design</h2> | ||
+ | <div class="col1 padBot3"> | ||
+ | <div class="col4 magRight2"> | ||
+ | <h3><a class="_link3" href="#!/pageModule1Assembly">Module 1 - PhotoCommunication Assembly/Design</a></h3> | ||
+ | <p>For the installation of an interaction system which is independent ... <a class="_link3" href="#!/pageModule1Assembly">Read More</a></p> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | <div class="col1"> | ||
+ | <div class="col4 magRight2"> | ||
+ | <h3><a class="_link3" href="#!/pageModule2Assembly">Module 2 - MurderCommunication Assembly/Design</a></h3> | ||
+ | <p>In view of the enormous problems we had with the cloning of Cph8 parts ... <a class="_link3" href="#!/pageModule2Assembly">Read More</a></p> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li id="pageSafetyVideo"> | ||
+ | <div class="box"> | ||
+ | <div class="closePlane"> | ||
+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | <div class="containerContent"> | ||
+ | <h2>Safety</h2> | ||
+ | <div class="col1"> | ||
+ | <div class="Btns1"> | ||
+ | <a href="#" class="upBtn"></a> | ||
+ | <a href="#" class="downBtn"></a> | ||
+ | </div> | ||
+ | <div class="scroll1"> | ||
+ | <object width="560" height="315"><param name="movie" value="http://www.youtube-nocookie.com/v/JUZbXYLhFlY?hl=tr_TR&version=3&rel=0"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube-nocookie.com/v/JUZbXYLhFlY?hl=tr_TR&version=3&rel=0" type="application/x-shockwave-flash" width="560" height="315" allowscriptaccess="always" allowfullscreen="true"></embed></object> | ||
+ | <p class="padBot4"><b>1. Would any of your project ideas raise safety issues in terms of:</b></p> | ||
+ | <p class="padBot4"><b>a. Researcher safety:</b></p> | ||
+ | <p class="padBot4">Actually few hazardous chemicals and solutions such as EtBr are used in some lab procedures such as gel preparation and electrophoresis. However these chemicals and solutions are used according to the safety rules of the laboratory with care and caution. All the members were trained for safety regulations of the laboratory as well as toxicity of the chemicals and solutions before starting the current project. </p> | ||
+ | <p class="padBot4"><b>b. Public Safety:</b></p> | ||
+ | <p class="padBot4">When released by accident, our parts and materials actually cause no damage to the general public. Due to the unability of E.coli strains, TOP10 and BL21 to survive out of the lab, they cannot pose any risk to the safety and health of the general public. </p> | ||
+ | <p class="padBot4"><b>c. Environment:</b></p> | ||
+ | <p class="padBot4">E. Coli strains TOP10 and BL1 have very limited ability to survive outside the laboratory; so that, it would be unable to survive or disseminate. Therefore, there is no specific environmental risk associated with the E. coli strains. All bacterial wastes are kept in 10% bleaching solution for one day, and then, are autoclaved to be sterilized. Yet, undesired GMOs may achieve ecologically harmful features. </p> | ||
+ | <p class="padBot4"><b>2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?</b></p> | ||
+ | <p class="padBot4">Solely, possible GMOs (genetically modified organisms) which are produced unwillingly during the lab procedures may cause unforeseen and incalculable consequences.A specific part of our Project ‘’MurderCommunication’’ is based on the prevention of GDOs. It contains quorum sensing dependent bacterial lysis. The cell wall destruction system consists of endolysin and holin proteins which are responsible for the lysis of the bacterium cell by degrading its cell wall. </p> | ||
+ | <p class="padBot4"><b>3. Is there a local biosafety group, committee, or review board at your institution?</b></p> | ||
+ | <p class="padBot4">The institution which we are using for laboratory facilities has its own biosafety rules. Rules for laboratory use, general principles, prevention from hazardous materials and application of emergency intervention in case of accident are included. The web link is given as (http://www.fatihmed.edu.tr/icerik/guvenlikkilavuzu.php). In Turgut Ozal University Medical School, Laboratory and Patient-Employee Safety Committee is responsible for control as well as biosafety of laboratories and safety of patients and employees. This committee works under one of the vice medical director of Turgut Ozal University Hospital, Prof. Dr. Mehmet Gunduz. Form on safety rules of Turgut Ozal University Medical School Laboratory use was filled in as required. We discussed our project with Prof. Dr. Mehmet Gunduz. Safety and security issues are found sufficient enough that no change is considered as necessary. (Further questions can be directed to Prof. Gunduz, tel: +90-312-203 5103, mgunduz@fatih.edu.tr) </p> | ||
+ | <p class="padBot4">Our advisor provided us with biosafety and lab training before starting our project. In the training, general safety rules of laboratory use, prevention from hazardous chemicals and solutions as well as emergency intervention in case of accident were included such as:</p> | ||
+ | <p class="padBot4">- Eating, drinking, storing food and smoking are absolutely not allowed.</p> | ||
+ | <p class="padBot4">- Mouth pipetting is not allowed; modern pipettes are used in lab.</p> | ||
+ | <p class="padBot4">- Hazardous wastes and ordinary wastes are separated and cautiously disposed.</p> | ||
+ | <p class="padBot4">- Lab coats are obligatory to wear in the lab and during on-going experiments.</p> | ||
+ | <p class="padBot4">- In electrophoresis room, lab coat, protective eyewear, lab masks and gloves have to be worn. In the case of use of EtBr; extra caution is required.</p> | ||
+ | <p class="padBot4">- Washing hands after any experiment and after touching anything related with viable material is obliged.</p> | ||
+ | <p class="padBot4">- Air conditioning is kept closed during on-going experiments in order to avoid possible infections of spores and bacteria.</p> | ||
+ | |||
+ | <p class="padBot4">In addition, we have made an instructive video about lab safety instuctions.</p> | ||
+ | |||
+ | <p class="padBot4">Turkey has national biosafety regulations and the link is given as http://www.tbbdm.gov.tr/Home/BioSafetyCouncilHome/BioSafetyCouncilHomeChoose.aspx. </p> | ||
+ | <p class="padBot4"><b>4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</b></p> | ||
+ | <p class="padBot4">- First, IGEM committee may prepare a lab safety acknowledgement form like OSHA form. This form can be required to be filled in by all members. This will allow us to confirm whether members of the teams are informed about safety issues and standardize the safety measurements.</p> | ||
+ | <p class="padBot4">- Second, iGEM committee may organize a webinar to inform the participants about safety and security in labs. Webinar can be done at the beginning of the experiments. Mentor scientists may talk about their experiences and mention some tricks about safety issues.</p> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li id="pageProjectOverview"> | ||
+ | <div class="box"> | ||
+ | |||
+ | <div class="closePlane"> | ||
+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | |||
+ | <div class="containerContent"> | ||
+ | <h2>Overview</h2> | ||
+ | <div class="col1 padBot3"> | ||
+ | <div class="col4 magRight2"> | ||
+ | <h3><a class="_link3" href="#!/pageModule1Overview">Module 1 - PhotoCommunication Overview</a></h3> | ||
+ | <p>The communication between bacteria is provided by chemicals like ... <a class="_link3" href="#!/pageModule1Overview">Read More</a></p> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | <div class="col1"> | ||
+ | <div class="col4 magRight2"> | ||
+ | <h3><a class="_link3" href="#!/pageModule2Overview">Module 2 - MurderCommunication Overview</a></h3> | ||
+ | <p>Bacteria communicate among each other by using chemicals called ... <a class="_link3" href="#!/pageModule2Overview">Read More</a></p> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li id="pageProjectGoals"> | ||
+ | <div class="box"> | ||
+ | <div class="closePlane"> | ||
+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | <div class="containerContent"> | ||
+ | <h2>Goals</h2> | ||
+ | <div class="col1"> | ||
+ | <div class="Btns1"> | ||
+ | <a href="#" class="upBtn"></a> | ||
+ | <a href="#" class="downBtn"></a> | ||
+ | </div> | ||
+ | <div class="scroll1"> | ||
+ | |||
+ | <p class="padBot4">Module1:PhotoCommunication & Module2:MurderCommunication – Goals</p> | ||
+ | <p class="padBot4"> <b>Achievement of communication between colonies which exist in different environments:</b></p> | ||
+ | <p class="padBot4">The communication between bacteria is provided by chemicals like AHL(N-Acyl Homoserine Lactones) in gram(-) bacteria, oligopeptids in gram(+) bacteria and some other molecules. These ways of messaging is possible only if both – sender and receiver of the message – are in the same medium, while messaging between two different media is not possible which is a limiting factor. As a solution for this standing problem and an alternative quorum sensing mechanism, ‘’Photocommunication’’ provides a non-chemical and contactless way of messaging between organisms.</p> | ||
+ | |||
+ | <p class="padBot4"> <b>Achievement of communication between different types of bacterial strains:</b></p> | ||
+ | <p class="padBot4">Between some types and subtypes of bacterial strains communication is already possible by means of different chemical molecules. However, it is known that not all types of bacteria have this ability. These microorganisms need alternative ways of interacting where ‘’photocommunication’’ comes into play. </p> | ||
+ | |||
+ | <p class="padBot4"> <b>Achievement of an new controlable prevention method against contamination:</b></p> | ||
+ | <p class="padBot4">Even if we use antibiotics in order to prevent the contamination in solid or liquid growth media, it is still not 100% safe against contamination. Our purpose is to use a second different antibiotic resistance for eliminating contamination as far as possible. The bacteria which we use in our exeriments will be able to survive by activating a second resistance at bright places thanks to the Cph8 photoreceptor system while unwanted bacteria die because of the lack of resistance against the second antibiotic. </p> | ||
+ | |||
+ | <p class="padBot4"> <b>Controlled inacivation/killing of the bacteria:</b></p> | ||
+ | <p class="padBot4">They same controlable system as mentioned above can also be used for avtivation and killing bacteria at a desired time. This time, the light source which had activated the second antibiotic resistance will be disabled so that bacteria which have done their jobs will be killed because of an antibiotic in their medium which they can’t resist any more. Thus, a processes can be terminated at each phase of the project, whenever desired -and more important- all this in a clear GDO-free manner.</p> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li id="pageModule1Overview"> | ||
+ | <div class="box"> | ||
+ | <div class="closePlane"> | ||
+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | <div class="containerContent"> | ||
+ | <h2 style="font-size:32px">PhotoCommunication - Overview</h2> | ||
+ | <div class="col1"> | ||
+ | <div class="Btns1"> | ||
+ | <a href="#" class="upBtn"></a> | ||
+ | <a href="#" class="downBtn"></a> | ||
+ | </div> | ||
+ | <div class="scroll1"> | ||
+ | |||
+ | <p class="padBot4">PhotoCommunication – Overview</p> | ||
+ | <p class="padBot4">The communication between bacteria is provided by chemicals like AHL(N-Acyl Homoserine Lactones) in gram(-) bacteria, oligopeptids in gram(+) bacteria and some other molecules. These ways of messaging is possible only if both – sender and receiver of the message – are in the same medium, while messaging between two different media is not possible which is a limiting factor.</p> | ||
+ | <p class="padBot4">In our project, we aimed to provide a solution for this problem and an alternative way for bacterial communication by using the light-contollable Cph8 photoreceptor system which was used quite early in the iGEM history (2004 Team UT Austin with the ‘’photographer bacteria’’) and called our project ‘’PHOTOCOMMUNICATION’’. Through the mechanism we have developed our bacteria(receiver) will be able to perceive the light that is produced by another bacteria(sender) and as an answer a further pathway in the receiver will be activated.</p> | ||
+ | <p class="padBot4">We prefered to work with E.coli in our project and tested two systems which will be applicable in different areas. In our first system (A), Cph8 photoreceptor was used to get an answer signal to the Cph8 induction in form of another Luciferase product or GFP. and in the second system (B) to develop an additional antibiotic resistance.</p> | ||
+ | <p class="padBot4">In system (A), our goal was to confirm the proper function of Cph8. The red light which was produced by the sender through the enzyme Red Luciferase activates the receiver Cph8 photoreceptor. After receiving the photons, EnvZ membrane protein which is anchored to Cph8 will phosphorylate another protein called OmpR at the inner side of the bacterial cell. Subsequently, OmpR will activate the pompC promoter and the sender bacteria will beginn to emit another luciferase product as an answer. Consequently, this would prove that the ‘’photocommunicaiton’’ mechanism has worked.</p> | ||
+ | <p class="padBot4">Our aim in system (B) was to use the CpH8 photoreceptor in order to earn the receiver bacteria an extra antibiotic resistance (apart from the one in the backbone) or, the other way around, to ensure the death of the receiver type bacteria by disrupting the connection at any desired time with the light/photon source so Cph8 can’t receive any activator signals any more and the bacteria loses it’s extra resistance placed behind the pOmpC promoter and is no more able to resist the second antibiotic in the medium. </p> | ||
+ | <p class="padBot4">The part sequences of system (A) and system (B) are identical from the pBAD promoter and pOmpC promoter. The differance between the both systems is the sequence after pOmpC. In (A) pOmpC is followed by Luciferase in 5’ direction while in (B) it is followed by the Ampicillin resistance gene.</p> | ||
+ | <p class="padBot4">We have shown the bacterial communication system we have designed via two different products. We think that by changing the sequence in the upstream region of the pOmpC promoter, it is possible to control divers pathways through an interbacterial communication. </p> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li id="pageModule2Overview"> | ||
+ | <div class="box"> | ||
+ | <div class="closePlane"> | ||
+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | <div class="containerContent"> | ||
+ | <h2 style="font-size:32px">Murder Communication - Overview</h2> | ||
+ | <div class="col1"> | ||
+ | <div class="Btns1"> | ||
+ | <a href="#" class="upBtn"></a> | ||
+ | <a href="#" class="downBtn"></a> | ||
+ | </div> | ||
+ | <div class="scroll1"> | ||
+ | |||
+ | <p class="padBot4">Bacteria communicate among each other by using chemicals called autoinducers. As a result, features like symbiosis, virulence, motility, antibiotic production and biofilm formation are modified at the gene expression level. This type of communication – called quorum sensing – can be amoung the same colony as well as between gram(-) and gram(+) bacteria. Regarding our former work, we decided to continue with communication between bacteria and to use quorum sensing molecules produced by the gram(-) bacteria P. aeruginosa and a similiar autoinducer chemicals like LuxI/R from another gram(-) bacteria V. Fischer - LuxI/R. [1][2]</p> | ||
+ | <p class="padBot4">This time, we thought of a innovative lysis device, similiar to that of the safety part of ‘’photocommunication’’. Contrary to ‘’Photocommunicaiton’’, bacteria will send each other chemical inducers instead of photons.</p> | ||
+ | <p class="padBot4">As a conclusion, we built a killing device wich is still related to bacterial quorum sensing mechanisms but additionally inducible with IPTG. We think that IPTG induction can serve as a control during the experiments on different colonies in the same media.</p> | ||
+ | <p class="padBot4">1..^ a b Passador, L.; Cook, J.M., Gambello, M.J., Rust, L., and Iglewski, B.H (1993). "Expression of Pseudomonas aeruginosa virulence genes requires cell-cell communication.". Science 260: 1127–1130. doi:10.1126/science.8493556. PMID 8493556.</p> | ||
+ | <p class="padBot4">2.^ a b Brint, J.M.; Ohman, D.E. (1995). "Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RhlR-RhlI, another set of regulators in strain PAO1 with homology to the autoinducer responsive LuxR-LuxI family.". J. Bacteriol. 177 (24): 7155–7163. PMC 177595. PMID 8522523</p> | ||
+ | |||
+ | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
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<div class="scroll1"> | <div class="scroll1"> | ||
- | <p class="padBot4"> | + | <p class="padBot4">In order to explain our project and SynBio better, we designed a game, which consists of the parts used in our project. The game based on famous card game, UNO. We modified some rules, but they are almost the same. Here are the cards and various descriptions:</p> |
- | + | ||
- | + | <p class="padBot4">The aim of the game is to get rid of all your cards. You need to play your cards when it is your turn. You have two choices. You can play the cards, which has the same type (colour). If you have not got the card which has the same colour with the card on the ground, you can play a card which forms the systems described. It is just needed your card to be come after the right part.</p> | |
- | + | ||
- | + | <p class="padBot4">If you have got the same card which is on the ground, you can play it whether it is your turn by saying “stop”.</p> | |
+ | |||
+ | <p class="padBot4">Red cards are terminators</p> | ||
+ | |||
+ | <p class="padBot4">Green card are promoters</p> | ||
+ | |||
+ | <p class="padBot4">Yellow cards are RBSs</p> | ||
+ | |||
+ | <p class="padBot4">Blue cards are protein sequences</p> | ||
+ | |||
+ | <p class="padBot4">NGSS cards, change the colour on the ground according to user’s preferences</p> | ||
+ | |||
+ | <p class="padBot4">iGEM cards, change the cards holder according to clock way</p> | ||
+ | |||
+ | <p class="padBot4">+2 cards, If you have got a +2 card, the person comes after you have to pick up two cards from the card stock. If the ones comes after you also have got one, this person play her / his card and it becomes to one after her / his. In these cases, last person have to pick up the sum of the cards on the ground.</p> | ||
+ | |||
+ | <p class="padBot4">+4 cards, have got the same role with +2 card, but the person can change the colour who plays the card latest.</p> | ||
+ | |||
+ | <p class="padBot4">Stop cards, stop that who is after you.</p> | ||
+ | |||
+ | <p class="padBot4">Turn cards, change the way of the game.</p> | ||
+ | <p class="padBot4"> </p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/2013hs/d/d4/Light_Responsive_System.png" width="500px" alt=""></div> | ||
+ | <p class="padBot4"> </p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/2013hs/4/4e/Right_Regulator_System_pOmpC_GFP.png" width="500px" alt=""></div> | ||
+ | <p class="padBot4"> </p> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/2013hs/0/02/2879_bp.png" width="500px" alt=""></div> | ||
+ | <p class="padBot4"> </p><p class="padBot4"> </p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <td><div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/a/ae/Blue.jpg" width="200px" alt=""></div></td></td> | ||
+ | <td> </td> | ||
+ | <td><div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/0/0e/Green.jpg" width="200px" alt=""></div></td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/4/45/Igemchange.jpg" width="200px" alt=""></div></td></td> | ||
+ | <td> </td> | ||
+ | <td><div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/0/0f/Ngsscolour.jpg" width="200px" alt=""></div></td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/b/b6/Plusfour.jpg" width="200px" alt=""></div></td></td> | ||
+ | <td> </td> | ||
+ | <td><div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/a/a2/Plustwo.jpg" width="200px" alt=""></div></td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/5/5a/Red.jpg" width="200px" alt=""></div></td></td> | ||
+ | <td> </td> | ||
+ | <td><div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/b/b4/Stop.jpg" width="200px" alt=""></div></td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/d/d8/Turn.jpg" width="200px" alt=""></div></td></td> | ||
+ | <td> </td> | ||
+ | <td><div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/7/7d/Yellow.jpg" width="200px" alt=""></div></td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
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<p class="padBot4">To present SynBio and İgem we have contacted with several companies. After couple of struggles we managed to start a communication with some of them. We prepared a stable presentation including SynBio, iGEM, Example Projects and Our Team.</p> | <p class="padBot4">To present SynBio and İgem we have contacted with several companies. After couple of struggles we managed to start a communication with some of them. We prepared a stable presentation including SynBio, iGEM, Example Projects and Our Team.</p> | ||
<p class="padBot4"><b>Energy Market Regulatory Authory</b></p> | <p class="padBot4"><b>Energy Market Regulatory Authory</b></p> | ||
- | <p class="padBot4">We have contacted with | + | <p class="padBot4">We have contacted with Osman Birgin, who is the head of Energy Market Regulatory Authory, he was enthusiastic for such kind of activities, so he has accepted our offer. We have taken appointment from him for 5th of June 2013 , at 11.00 am. Mariye Erol and Dilara Soylu as the students and Fatma Betül Çevik as the advisor have gone for meeting. When we arrived, we had a conversation in Osman Birgin’s room. Afterward we passed to meeting room and started our presentation. The presenatation has been done to Omer Birgin, other department presidents and a septet of EMRA workers. </p> |
- | <p class="padBot4">The audience has listened the presentation of little-known discipline synthetic biology and iGEM competition carefully. We’ve informed them about the studies on İgem related to energy. Our friends have answered the questions. Then we talked about the future of SynBio. | + | <p class="padBot4">The audience has listened the presentation of little-known discipline synthetic biology and iGEM competition carefully. We’ve informed them about the studies on İgem related to energy. Our friends have answered the questions. Then we talked about the future of SynBio. Osman Birgin offered us sending our presentation documents as an email to the ones on their email lists who are almost five hundred people.</p> |
- | <p class="padBot4">We informed them about our outreach projects. After couple of time, | + | <p class="padBot4">We informed them about our outreach projects. After couple of time, Osman Birgin offered to us sending our presentation document as email to the ones on their email list who are almost five hundred people (Document was helpful :)) We have left EMRA about 12.40 am. Beyond being able to reach our purposes, we enjoyed to spend time with them!</p> |
<table> | <table> | ||
<tr> | <tr> | ||
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</table> | </table> | ||
<p class="padBot4"><b>Halıcızade Company</b></p> | <p class="padBot4"><b>Halıcızade Company</b></p> | ||
- | <p class="padBot4">From our team; Mariye Erol, Dilara Soylu and Fatma Betül Çevik have established communication with the founder and the head of Halicızade Company, | + | <p class="padBot4">From our team; Mariye Erol, Dilara Soylu and Fatma Betül Çevik have established communication with the founder and the head of Halicızade Company, Metin Özer. We have talked about whether we can make a presentation or not Metin Ozer has accepted our offer.</p> |
- | <p class="padBot4">The presentation has been placed on 5 June 2013, at 14.00. It has lasted for 30 minutes. Firstly we explained what SynBio and İgem is. Then we have showed the works of previous teams. After we completed the presentation, they spent some time on possible kinds of projects that can be done in the area of SynBio. It was a beneficial presentation for us and him, | + | <p class="padBot4">The presentation has been placed on 5 June 2013, at 14.00. It has lasted for 30 minutes. Firstly we explained what SynBio and İgem is. Then we have showed the works of previous teams. After we completed the presentation, they spent some time on possible kinds of projects that can be done in the area of SynBio. It was a beneficial presentation for us and him, Metin Ozer has given his thanks. Afterward we left him our presentation and sponsorhip document and left Halıcızade Company at 14.00. </p> |
<p/><p/><p> Below you can find some photos from the our presentations </p> | <p/><p/><p> Below you can find some photos from the our presentations </p> | ||
<table> | <table> | ||
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</table> | </table> | ||
<p class="padBot4"><b>FIRAT COMPANY</b></p> | <p class="padBot4"><b>FIRAT COMPANY</b></p> | ||
- | <p class="padBot4"> | + | <p class="padBot4">Metin Fırat has accepted our presentation offer kindly. On 18 May, at 10.00 a.m. Sinem and Hatice from our team went to Fırat Company for the presentation. Audience consisted of six person. We used the powerpoint slides to inform the listeners. After sincere conversation we left the company. It was a succesfull presentation, we also developed our presentation skills.</p> |
<p/><p/><p> Below you can find some photos from the our presentations </p> | <p/><p/><p> Below you can find some photos from the our presentations </p> | ||
<table> | <table> | ||
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</div> | </div> | ||
</li> | </li> | ||
+ | |||
+ | <li id="pageFirstWeek"> | ||
+ | <div class="box"> | ||
+ | <div class="closePlane"> | ||
+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | <div class="containerContent"> | ||
+ | <h2>First Week of June</h2> | ||
+ | <div class="col1"> | ||
+ | <div class="Btns1"> | ||
+ | <a href="#" class="upBtn"></a> | ||
+ | <a href="#" class="downBtn"></a> | ||
+ | </div> | ||
+ | <div class="scroll1"> | ||
+ | |||
+ | <p class="padBot4">06.01.2013 Saturday</p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • We have transformed Luciferase, Red Luciferase,EPIC, pOmpR, pLac, RFP (Amp),Rzt, pOmpR+GFP, Cph8. (Tomorrow will taken at 10.40 am)</li> | ||
+ | <li> • We have talked to Gökhan Nas for the pirimers which are necessery for Gibson.</li> | ||
+ | <li> • 4 plates with Amp and the whole lab has been prepared.</li> | ||
+ | <li> • The plates wih Chl(1) and AK(5) used from which has iGEM sent.</li> | ||
+ | <li> • For AC and Amp, we used which we have prepared.</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <p class="padBot4">06.02.2013 Sunday</p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • Only EPIC(1),Luc(1), pLac(1), pOmpR+GFP(3), RLuc(1) colonies grew</li> | ||
+ | <li> • We thought that the problem could be over antibiotic sowe have transformed full 9 genes with 0.5 Chl and 0.75 Amp again. (tomorrow will taken at 2.20 am)</li> | ||
+ | <li> • 0.5 Chl and 0.75 Amp plates have prepared and mot of them used.</li> | ||
+ | <li> • For Cph8, we did two different examples on Amp and Chl plates. The AC plates that we have are not useful.</li> | ||
+ | <li> • For AK (Rzt), we have made two dfferent examples from 12.12 date plates and iGEM plates</li> | ||
+ | <li> • From Saturday: EPIC, Luc, pLac, pOmpR+GFP,RLuc examples’ liquid cultures have done. The isolation will be on tomorrow with team.</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <p class="padBot4">06.03.2013 Monday </p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • The kit of plasmid isolation is ready.</li> | ||
+ | <li> • EPIC,3,RLoo,Loo has isolated.</li> | ||
+ | <li> • EPIC and 3 have cut from Xp; Loo and RLoo from ES but we have failed.</li> | ||
+ | <li> • The liquid cultures from tomorrow have isolated.(EPIC,3,6,9,Loo,8)</li> | ||
+ | |||
+ | <li> pLac: 9</li> | ||
+ | <li> Luciferase: Loo</li> | ||
+ | <li> Red luc: RLoo</li> | ||
+ | <li> EPIC: EPIC</li> | ||
+ | <li> RFP: RFP</li> | ||
+ | <li> Cph8: 8</li> | ||
+ | <li> pOmpC: 6</li> | ||
+ | <li> RBS+LacZ+term: π</li> | ||
+ | <li> pOmpC+RBS+GFP: 3</li> | ||
+ | <li> The parts that we are going to make: 9EPIC, 83, 6π, 86π</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <p class="padBot4">06.04.2013 Tuesday </p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • PCR of 3,6,9,8 have done.</li> | ||
+ | <li> • 8 and EPIC’s electrophoresis results are abortive.</li> | ||
+ | <li> • Loo and RLoo transformed.</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <p class="padBot4">06.05.2013 Wednesday</p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • 8 and EPIC PCR again. (2.30 pm)</li> | ||
+ | <li> • 8 and EPIC electrophoreis results are abortive.</li> | ||
+ | <li> • 8 and EPIC PCR again(4.00 pm)</li> | ||
+ | <li> • RLoo transformation is succesful but Loo is not.</li> | ||
+ | <li> • Loo,RLoo transformated. (we will get at 01.05 am)</li> | ||
+ | <li> • RLoo liquid culture has done. (we will get at 00.20 am)</li> | ||
+ | <li> • 8,EPIC,RFP transformated. (tomorrow at 8.40 am)</li> | ||
+ | <li> • 8 and EPIC electrophoresis results are abortive again.</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/d/da/05.06.13_PCR_%288%2C_EPIC%2C_mix%29kesik.png" width="400" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/b/b6/05.06.13_PCR_%28EPIC-8%29kesik.png" width="400" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/b/b6/05.06.13_PCR_%28EPIC-8%29kesik.png" width="400" alt=""></div> | ||
+ | |||
+ | <p class="padBot4">06.06.2013 Thursday</p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • The liquid culture of Loo and RLoo which we got at 1.05 am have done.(will come out at 1.30 am)</li> | ||
+ | <li> • The RLoo from 1.20 am have isolated.</li> | ||
+ | <li> • 8 has transformed. (will come out at 9.00 am)</li> | ||
+ | <li> • The digestion and electrophoresis of 3,6,9 from PCR; RLoo(1),RLoo(2), RLoo(3),EPIC(2), EPIC(3),3(2), 9(3) from isolation have done. 6,9,9(3) are unsuccesful because of the problem with gel. RLoo(1), RLoo(2), RLoo(2) are unsuccesful. 3 and 3(2) are succesful. EPIC is suspecious.</li> | ||
+ | <li> • EPIC’s liquid culture have done.(will come out at 8.30 am)</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/6/6f/06.06.13_dig_b_kesik.png" width="400" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/9/99/06.06.13_pcr-iso_dig_kesik.png" width="400" alt=""></div> | ||
+ | |||
+ | <p class="padBot4">06.07.2013 Friday </p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • The electrophoresis of 6,9 from PCR and 9(3) from isolation have done. Two gels have prepared.(%1 and %2) (80 V 40 mins.)</li> | ||
+ | <li> • %2 gel failed.</li> | ||
+ | <li> • %1 gel is succesful; 6 and 9 are able to use.</li> | ||
+ | <li> • The RLoo and loo from 1.30 am have isolated.</li> | ||
+ | <li> • Loo(2), Loo(3),RLoo(1), RLoo(2) and RLoo(3) have isolated.</li> | ||
+ | <li> • Loo(2), Loo(3),RLoo(1), RLoo(2), RLoo(3), EPIC(1),EPIC(2) and EPIC(3) have digested. EPIC(2)’s electrophoresis result is succesful but EPIC(1) and EPIC(3) ares suspecious.</li> | ||
+ | <li> • Loo(2), Loo(3),RLoo(1), RLoo(2) and RLoo(3) are unsuccesful.</li> | ||
+ | <li> • Loo and RLoo’s PCR have done.</li> | ||
+ | <li> • Loo and RLoo’s PCR results’ electrophoresis have done and we have failed again.</li> | ||
+ | <li> • Loo,RLoo,3(3),500 are digested.</li> | ||
+ | <li> • 8’s liquid culture have done(will come out 8.00 am)</li> | ||
+ | <li> • Loo and RLoo’s electrophoresis have failed again!</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/5/50/07.06.13_ISO_Dig_kesik.png" width="400" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/a/af/07.06.13_pcr_b_kesik.png" width="400" alt=""></div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li id="pageSecondWeek"> | ||
+ | <div class="box"> | ||
+ | <div class="closePlane"> | ||
+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | <div class="containerContent"> | ||
+ | <h2>Second Week of June</h2> | ||
+ | <div class="col1"> | ||
+ | <div class="Btns1"> | ||
+ | <a href="#" class="upBtn"></a> | ||
+ | <a href="#" class="downBtn"></a> | ||
+ | </div> | ||
+ | <div class="scroll1"> | ||
+ | <p class="padBot4"></p> | ||
+ | <div class="picHold"><img src="" width="" alt=""></div> | ||
+ | <p class="padBot4">06.08.2013 Saturday</p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • 8’s liquid culture have failed.</li> | ||
+ | <li> • Loo , RLoo and 3(3)’s digestion examples’ electrophoresis have done.</li> | ||
+ | <li> • Loo and RLoo’s bands are not on the right places.</li> | ||
+ | <li> • 3(3) had not cut.</li> | ||
+ | <li> • 500 is succesful.</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/1/1b/08.06.13_dig_kesik.png" width="400" alt=""></div> | ||
+ | <p class="padBot4">06.09.2013 Sunday</p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • The liquid cultures of 6-EPIC 1, 6-EPIC 2, 9-EPIC 1, 9EPIC 2 have done which came out 5.30 am and 7.10. (The liquid culture A will be on 20.15 and B will be on 22.45)</li> | ||
+ | <li> • 8 has transformated.(will be on 1.10) It has done with 5 different antibiotic resistanced plates (amp, chl, AC,AK,KAN)</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <p class="padBot4">06.10.2013 Monday</p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • The 8 plates went out at 01.15 am. (threee plates have nothing,a plate (chl) have 3 colonies and another plate (amp) have a colonie.</li> | ||
+ | <li> • All of the 4 colonies' liquid culture have done. (8-amp, 8-chl 1, 8-chl 2, 8-chl 3)</li> | ||
+ | <li> • A(20.15) and B(22.25) liquid cultures have isolated. The results are fine.</li> | ||
+ | <li> • Isolated and after that digested.</li> | ||
+ | <li>i. 6 EPIC A 2/1 </li> | ||
+ | <li>ii. 6 EPIC A 2/2</li> | ||
+ | <li>iii. 6 EPIC B 1/1 6EPIC's- Xba1+Pst1</li> | ||
+ | <li>iv. 6 EPIC B 1/1 9EPIC's-EcoR1+Pst1 (have cut with)</li> | ||
+ | <li>v. 6 EPIC B 2/1</li> | ||
+ | <li>vi. 9 EPIC A 1/1</li> | ||
+ | <li>vii. 9 EPIC A 1/2</li> | ||
+ | <li>viii. 9 EPIC A 2/2</li> | ||
+ | <li> • Electrophoresis have done. The examples have walked a little.</li> | ||
+ | <li> • Electrophoresis of the same examples have done again.</li> | ||
+ | <li> • Two competent cells have taken from AUC TURKEY team just for trying and CpH8's and Red Firefly Luciferase's (lc325239) transformation have done. (will have at 07.15 pm)</li> | ||
+ | <li> • RLoo and Loo's liquid cultures have put to the ETÜV little bit opened. (will be at 19.40 pm)</li> | ||
+ | <li> • 6 EPIC A 2/1 ,6 EPIC A 2/2,6 EPIC B 1/1, 6 EPIC B 1/1, 6 EPIC B 2/1,9 EPIC A 1/1, 9 EPIC A 1/2, 9 EPIC A 2/2 have digested again.</li> | ||
+ | <li> • The resultant RLoo and CpH8's (7.15 pm) liquid cultures have done. (will be at 11.45 am) (5 Loo, 4 RLoo, 4 CpH8)</li> | ||
+ | <li> • Loo, RLoo and CpH8 have isolated.</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <p class="padBot4">06.11.2013 Tuesday</p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • The isolation results are normal. CpH8s are goingto digest from EcoR1- Sp.</li> | ||
+ | <li> • Long PCR have done for 8, EPIC and 500 Gibson A.</li> | ||
+ | <li> • The Gibson primers have arrived for 8, EPIC and 6. diluted with TeBuffer.</li> | ||
+ | <li> • 500 have diluted from the kit. The nucleic asid amount have measured on Nanodrop.</li> | ||
+ | <li> • 6 EPIC and 9 EPIC's electrophoresis have done again. 6 EPIC is succeed but 9 EPIC failed. (01.00 am)</li> | ||
+ | <li> • 1C3, EPIC and 8's PCR products' electrophoresis have done. 1C3 is true the others have not got band.</li> | ||
+ | <li> • 8Z's transformation have done. (will be at 01.05 pm)</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/7/7d/11.06.13_dig_%288%29_kesik.png" width="400" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/4/4c/11.06.13_Lig_3._y%C3%BCr_kesik.png" width="400" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/2/26/11.06.13_Long_Pcr_kesik.png" width="400" alt=""></div> | ||
+ | |||
+ | <p class="padBot4">06.12.2013 Wednesday</p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • 8 GC, 8 HF, EPIC GC, EPIC HF's PCR and electrophoresis have done. EPIC is succeed but 8 isn't. 8 needs long PCR.</li> | ||
+ | <li> • 8, 8Z and RFP have transformated.(will be at 06.05 am)</li> | ||
+ | <li> • 8 and EPIC's digestion and electrophoresis have done but no display.</li> | ||
+ | <li> • We have waited arabinose but it didn't come.</li> | ||
+ | <li> • No colonies from 8Z.</li> | ||
+ | <li> • 7 Chl plates have prepared.</li> | ||
+ | <li> • To learn is there any problem with primers because of there's no band on long PCR, the primer electrophoresis have done.</li> | ||
+ | <li>I. The primers have walked on 250 V, 2.5 mins, %1 gel.</li> | ||
+ | <li>II. EPIC F, 8R and 6R were pale. Wen could not figure out where is the problem, putting or diluting. We will ask tomorrow.</li> | ||
+ | <li> • 8 has cut directly from BioBrick simple (5 µL) If the problem was on the band, it will dictly send and reported to iGEM.</li> | ||
+ | <li> • The BioBrick simples have finished so we have diluted from AUC TURKEY team.</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/8/80/12.06.13_8ep_dig_kesik.png" width="400" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/2/2e/12.06.13_High_Fidelity_PCR_kesik.png" width="400" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/b/bd/12.06.13_Primerler_kesik.png" width="400" alt=""></div> | ||
+ | |||
+ | <p class="padBot4">06.13.2013 Thursday</p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • 8's long PCR and 6 gradient's PCR have done again.</li> | ||
+ | <li> • 8's and 6's PCR's electrophoresis have done. 8 have failed again!!</li> | ||
+ | <li> • Colonies have chosen from Loo and RLoo and their liquid cultures have done again.</li> | ||
+ | <li> • We are going to continue with 64 fr</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/9/93/13.06.13_dig%2BPCR%288%29_kesik.png" width="400" alt=""></div> | ||
+ | <p class="padBot4">06.14.13 Friday</p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • HO1, CHO1k, R8, RZ's PCR have done but unsuccesful.</li> | ||
+ | <li> • With Omer Birgin's procedure.) HO1k, R8, 8 are transformated. (will be at 3.00.)</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/7/71/14.06.13_pcr_kesik.png" width="400" alt=""></div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li id="pageThirdWeek"> | ||
+ | <div class="box"> | ||
+ | <div class="closePlane"> | ||
+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | <div class="containerContent"> | ||
+ | <h2>Third Week of June</h2> | ||
+ | <div class="col1"> | ||
+ | <div class="Btns1"> | ||
+ | <a href="#" class="upBtn"></a> | ||
+ | <a href="#" class="downBtn"></a> | ||
+ | </div> | ||
+ | <div class="scroll1"> | ||
+ | <p class="padBot4"></p> | ||
+ | <div class="picHold"><img src="" width="" alt=""></div> | ||
+ | <p class="padBot4">06.15.13 Saturday</p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • 4, 5, 7, 9 have transformated. (will be at 05.30.) </li> | ||
+ | <li> • 7, 9's PCR results' (16-17. 09.12 date) and 7, 9 iso results' dig electrophoresis have done.</li> | ||
+ | <li>7 old, 9 old (12.09.12)</li> | ||
+ | <li>7 iso, 9 iso </li> | ||
+ | <li>9(17), 9(16)</li> | ||
+ | <li>7 green, 7 purple</li> | ||
+ | <li> • 9(17), 9(16), 7 green, 7 purple's electrophoresis have done again.</li> | ||
+ | <li> • 4, 5, 7, 9 electrophoresis have done again.</li> | ||
+ | <li> • 4, 5, 7, 9 PCR have done.PCR Pur done and digested.</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/e/ea/15.06.13_kesik.png" width="400" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/7/71/15.06.13_PCR_kesik.png" width="400" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/c/c9/15.06.13_pcr_pro_kesik.png" width="400" alt=""></div> | ||
+ | <p class="padBot4">06.16.13 Sunday</p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • 4, 5, 7, 9 ligested. (9-4, 5-7)</li> | ||
+ | <li> • 94, 57 transformated.</li> | ||
+ | <li> • On the transformation from 5.30 4, 5 is unsuccesful.</li> | ||
+ | <li> • 4, 5 transformated. (will be at 05.45 am)</li> | ||
+ | <li> • 5, after PCR Pur cut simple's electrophoresis have done. (100 bp, 75 V, 20 min)</li> | ||
+ | <li> • There is no colonies of 94,57 from 6.00pm.</li> | ||
+ | <li> • 94, 57 transformated again. (will be at 11.10 am.)</li> | ||
+ | <li> • 7, 9 liquid cultures have done19.50’de alındı.)</li> | ||
+ | <li> • 7, 9 isolated.</li> | ||
+ | <li> • pTetR PCR have done. Because of it has put into gel wrong the result is unsuccesful.</li> | ||
+ | <li> • pTetR PCR Pur have done.</li> | ||
+ | <li> • 7, 9 iso, pTetR PCR simples have digested.</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/5/51/16.06.13_pcr_kesik.png" width="400" alt=""></div> | ||
+ | <p class="padBot4">06.17.13 Monday</p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • 7, 9 iso, pTetR PCR dig simples' electrophoresis have done. 7, 9 succesful, pTetR unsuccesful(because of the gel.)</li> | ||
+ | <li> • pTetR electrophoresis have done two times again.(75 V-25 min) unsuccesful.</li> | ||
+ | <li> • pTetR have transformated. (will be at 8.40 pm.)</li> | ||
+ | <li> • Because of there is no colonies of 4 and 5 from 05.45 am, it has transformed again. (10.25 pm)</li> | ||
+ | <li> • 2012 pTetR iso simples have digested from ES. (75 V-25 min-10 bp) unsuccesful.</li> | ||
+ | <li> • 94 have ligested with 500 and transformated. (will be at 03.55 am.)</li> | ||
+ | <li> • Amp plates have prepared, two of them have given to AU</li> | ||
+ | <li> • 57 PCR, 57 iso simples' liquid cultures have done. (will be at 1.45 am)</li> | ||
+ | <li> • At 20.40, the pTetR plates have taken. Again there was empty bubles on it, just incase 3 liquidcultures have prepared.(will be at 11.00 am)</li> | ||
+ | <li> • Because of there were empty bubles in most plates, for controle the transformation:</li> | ||
+ | <li>i. From AUC, 2 NEB comp. have taken and a TOP 10 comp have taken from us.</li> | ||
+ | <li>ii. 594 done from NEB10. RFP Amp done from a NEB 10 and a TOP 10.</li> | ||
+ | <li>iii. There were not any Chl plates so we have borrowed from AUC, For 594 transformation plates are tomorrow will be at 2.55 pm.</li> | ||
+ | <li> • There were still not colonies of 4 and 5 from 10.45 pm. There were empty bublles in it so we did not do a liquid culture.</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/2/2e/17.06.13_dig%28iso%29_kesik.png" width="400" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/6/65/17.06.13_dig%28pTetR%292_kesik.png" width="400" alt=""></div> | ||
+ | |||
+ | <p class="padBot4">18.06.13 Tuesday</p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • 57 iso, PCR (1,2,3) simples have isolated.06.18.13 Tuesday</li> | ||
+ | <li> • 94's liquid cultures have done.(will be at 10.45 pm)</li> | ||
+ | <li> • 57 have digested, electrophoresis have done but results are unsuccesful.</li> | ||
+ | <li> • From 3.00 pm 94 plates' liquid cultures have done.</li> | ||
+ | <li> • 500, pTetR, pcons have digested.</li> | ||
+ | <li> • 57, pTetR 7,pcons7 have connected with 500 and linner backbones.</li> | ||
+ | <li> • From 10.45 pm isolation results' nanodrops have done.</li> | ||
+ | <li> • The resultant 3 colonies of 94 plates' second one have digested.</li> | ||
+ | <li> • CpH8 is trying again on antibiotic-free plates.(will be at 10.10 am)</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/3/31/18.06.13_dig_5_500_kesik.png" width="400" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/4/45/18.06.13_dig_57_kesik.png" width="400" alt=""></div> | ||
+ | <p class="padBot4">06.19.13 Wednesday</p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • 94 (2) have digested.</li> | ||
+ | <li> • 57,pTetR7,pcons 7 have transformated. (will be at 5.35 pm)the liquid cultures have done. (will be at 8.20 am)</li> | ||
+ | <li> • 94(2) electrophoresis have done, results are succesful.</li> | ||
+ | <li> • 94's (second cultures) have isolated, digested and electrophoresis done. The results are succesful.</li> | ||
+ | <li> • CpH8's liquid cultures have done. (will be at 3.23 am)</li> | ||
+ | <li> • pLac LasI, LasI GFP PCR have done. Electrophoresis results are unsuccesful.</li> | ||
+ | <li> • 13 Chl plates have prepared.</li> | ||
+ | <li> • The liquid cultures from 3.23 am have isolated. Because of the nanodrop results were not good, just 8 1b hae digested. electrophoresis have done but results were unsuccesful.pLacI LasI and LasI GFP liquid cultures have not prepared because there were not any colonies in the plate.</li> | ||
+ | <li> • LasI GFP have transformed again.(1 comp cell from NGSS, 1 comp cell from AUC)</li> | ||
+ | <li> • 57 (500), pTetR7 liner, pCons7 liner have digested, the electrophoresis have done but results are wrong, 57(500) electrophoresis have done again but there is no band. </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/b/bf/19.06.13_dig_94_%281-2-3%29_kesik.png" width="400" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/a/a9/19.06.13_dig_94_kesik.png" width="400" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/e/ef/19.06.13_pcr_kesik.png" width="400" alt=""></div> | ||
+ | <p class="padBot4">06.20.13 Thursday</p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • 13 Chl plates have prepared.</li> | ||
+ | <li> • The isolation of 03.23 am liquid cultures have done.</li> | ||
+ | <li> • Because of the nanodrop results was not good, just CpH8 1b have digested but it is unsuccesful.</li> | ||
+ | <li> • The liquid cultures of pLac, LasI, LasI(cfp) did not prepared because there were not any colonies.</li> | ||
+ | <li> • LasI(cfp) have transformated again. (1 compt. Cell NGSS, 1 compt. Cell AUC)</li> | ||
+ | <li> • 57(500), pTetR7(lineer), pcons7(lineer) have isolated. (The backbones were wrong except pTetR's.)</li> | ||
+ | <li> • 57, 7 pcons7 have digested. 57 have tried again. the others are unsuccesful.</li> | ||
+ | <li> • 57' electrophoresis have done again but there was not band.</li> | ||
+ | <li> • 507 have ligested.</li> | ||
+ | <li> • 507 have transformated. (will be at 8.00 am.)</li> | ||
+ | <li> • Gfp, LasI liquid cultures have done. (will be at 2.45 pm.)</li> | ||
+ | <li> • 594, 2, 2b, 1b's liquid cultures have prepared. (will be at 10.25 pm.)</li> | ||
+ | <li> • Ecfp and LasI’s colonies have grew yesterday transformation's liquid cultures so it has put into the same broth with 594 1b. Later on although the controle, we realize the transformation did not happen. </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/7/77/20.06.13_dig_57_kesik.png" width="400" alt=""></div> | ||
+ | <div class="picHold"><img src="https://static.igem.org/mediawiki/igem.org/9/9a/20.06.13_dig%28cons7%2C500%2C57%29_kesik.png" width="400" alt=""></div> | ||
+ | <p class="padBot4">06.21.13 Friday</p> | ||
+ | <div class="itemlist"> | ||
+ | <ul > | ||
+ | <li> • The liquid cultures of 08.00 am 507 plates have done. (will be at 10.30 pm.)</li> | ||
+ | <li> • LasI and CFP from 11.45 am have isolated and digested but the results are unsuccesful.</li> | ||
+ | <li> • 507 have isolated.</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | |||
+ | <li id="pageLabNotebook"> | ||
+ | <div class="box"> | ||
+ | |||
+ | <div class="closePlane"> | ||
+ | <a class="closeButton" href="#!/pageHome"><img src="https://static.igem.org/mediawiki/2013hs/5/57/CloseIcon.png" alt=""></a> | ||
+ | </div> | ||
+ | |||
+ | <div class="containerContent"> | ||
+ | <h2>Lab Notebook/June</h2> | ||
+ | <div class="col1 padBot3"> | ||
+ | <div class="col4 magRight2"> | ||
+ | <h3><a class="_link3" href="#!/pageFirstWeek">First week of June</a></h3> | ||
+ | <p>Notebook/First week</p> | ||
+ | |||
+ | </div> | ||
+ | <div class="col4"> | ||
+ | <h3><a class="_link3" href="#!/pageSecondWeek">Second week of June</a></h3> | ||
+ | <p>Notebook/Second week </p> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | <div class="col1"> | ||
+ | <div class="col4 magRight2"> | ||
+ | <h3><a class="_link3" href="#!/pageThirdWeek">Third week of June</a></h3> | ||
+ | <p>Notebook/Third week</p> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
<li id="pageComics"> | <li id="pageComics"> | ||
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</div> | </div> | ||
<div class="scroll1"> | <div class="scroll1"> | ||
- | <p class="padBot4"> | + | <p class="padBot4">We had a meeting with AUC Turkey Team on 18th Of June at 2.00 pm. In this meeting teams explained their project mutually. After Presentations teams shared their positive and negative ideas about projects.</p> |
+ | <p class="padBot4">We had material exchange among two teams. </p> | ||
+ | |||
+ | <p class="padBot4">Cph8 was very important for our project, but although trying every way, we haven't got a positive result. So, we used cph8 in kits of AUC Turkey and Fatih Medical team</p> | ||
+ | |||
+ | <p class="padBot4">We received K769001 and 10407 genes from ATOMS2013 team; K772004 and K772005 genes from Fatih Medical team</p> | ||
+ | <p class="padBot4">While uploading our wiki to main page, we had some minor problems. So we got some help from AUC_turkey team.</p> | ||
<p/><p/><p> Below you can find some photos </p> | <p/><p/><p> Below you can find some photos </p> | ||
<table> | <table> | ||
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<div class="scroll1"> | <div class="scroll1"> | ||
<p class="padBot4">One day of an iGEMer: "Experiment, wiki, experiment, wiki, experiment, wiki... zZzZzZz..." </p> | <p class="padBot4">One day of an iGEMer: "Experiment, wiki, experiment, wiki, experiment, wiki... zZzZzZz..." </p> | ||
+ | <p class="padBot4">Of course this is not true. We had lots of fun while we were studying. </p> | ||
<p class="padBot4">Listening Ayşe Sister's guitar was a great pleasure for us. Also Dilara can play, but... In our free times, it is not valid actually, we played table tennis or took videos. The best parts of the days, definitely, meal times. Beril's mother bought us a baklava (turkish dessert, yummy!) for once :) Our traditional meal is pizza. Ordering pizza is cheaper than the others. We can choose "Secrets" as our favourite team song. Our favourite sport is wrestling, also.After three weeks that we stayed in the lab, we have collected some funny moments. Beyond them we took some videos else too. Enjoy it!"</p> | <p class="padBot4">Listening Ayşe Sister's guitar was a great pleasure for us. Also Dilara can play, but... In our free times, it is not valid actually, we played table tennis or took videos. The best parts of the days, definitely, meal times. Beril's mother bought us a baklava (turkish dessert, yummy!) for once :) Our traditional meal is pizza. Ordering pizza is cheaper than the others. We can choose "Secrets" as our favourite team song. Our favourite sport is wrestling, also.After three weeks that we stayed in the lab, we have collected some funny moments. Beyond them we took some videos else too. Enjoy it!"</p> | ||
- | + | <object width="560" height="315"><param name="movie" value="http://www.youtube-nocookie.com/v/6BQHt_C7hN8?version=3&hl=tr_TR&rel=0"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube-nocookie.com/v/6BQHt_C7hN8?version=3&hl=tr_TR&rel=0" type="application/x-shockwave-flash" width="560" height="315" allowscriptaccess="always" allowfullscreen="true"></embed></object> | |
</div> | </div> | ||
</div> | </div> | ||
Line 1,076: | Line 2,192: | ||
<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>• Pcr Tube</li> | + | <li><li> • Pcr Tube</li> |
- | <li>• Pipette tip which has filter</li> | + | <li><li> • Pipette tip which has filter</li> |
- | <li>• Plasmid which had isolated</li> | + | <li><li> • Plasmid which had isolated</li> |
- | <li>• -20 C EcoR1, Spel, Pstl, Xbal enzymes</li> | + | <li><li> • -20 C EcoR1, Spel, Pstl, Xbal enzymes</li> |
- | <li>• ×10 NE Buffer 2</li> | + | <li><li> • ×10 NE Buffer 2</li> |
- | <li>• ×100 BSA</li> | + | <li><li> • ×100 BSA</li> |
- | <li>• Nuclease Free H2O</li> | + | <li><li> • Nuclease Free H2O</li> |
- | <li>• Heat block or PCR</li> | + | <li><li> • Heat block or PCR</li> |
</ul> | </ul> | ||
</div> | </div> | ||
Line 1,089: | Line 2,205: | ||
<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>• Take PCR tubes. Don’t forget to take tube for destination plasmid.</li> | + | <li><li> • Take PCR tubes. Don’t forget to take tube for destination plasmid.</li> |
- | <li>• Paste the stickers to the tubes.</li> | + | <li><li> • Paste the stickers to the tubes.</li> |
- | <li>• Lave NE Buffer 2, NF Water, BSA into ice when they melted.</li> | + | <li><li> • Lave NE Buffer 2, NF Water, BSA into ice when they melted.</li> |
- | <li>• Take the average of the nucleic acid concentrations measured by the spectrometer. Divide 500 by the DNA average.</li> | + | <li><li> • Take the average of the nucleic acid concentrations measured by the spectrometer. Divide 500 by the DNA average.</li> |
- | <li>• Add 5µl Ne Buffer.</li> | + | <li><li> • Add 5µl Ne Buffer.</li> |
- | <li>• Add 0.5µl BSA Buffer.</li> | + | <li><li> • Add 0.5µl BSA Buffer.</li> |
- | <li>• Add 1 µl of the enzymes with barrier tips.</li> | + | <li><li> • Add 1 µl of the enzymes with barrier tips.</li> |
- | <li>• If you cut with Ecor1 and SpI, it will be up stream.</li> | + | <li><li> • If you cut with Ecor1 and SpI, it will be up stream.</li> |
- | <li>• If you cut with Xbal and Pst1, it will be down stream.</li> | + | <li><li> • If you cut with Xbal and Pst1, it will be down stream.</li> |
- | <li>• Subtract the amount of DNA from 42.5 µl. This result will be the amount of NFW used.</li> | + | <li><li> • Subtract the amount of DNA from 42.5 µl. This result will be the amount of NFW used.</li> |
- | <li>• Add the NFW with barrier tips and do one pippetting while taking the NFW.</li> | + | <li><li> • Add the NFW with barrier tips and do one pippetting while taking the NFW.</li> |
- | <li>• Then the DNA is put into the PCR and is left there for 30 minutes.</li> | + | <li><li> • Then the DNA is put into the PCR and is left there for 30 minutes.</li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>• Be sure enzymes are enough.</li> | + | <li><li> • Be sure enzymes are enough.</li> |
- | <li>• You must use plasmid which had isolated.</li> | + | <li><li> • You must use plasmid which had isolated.</li> |
- | <li>• Use barrier tips has filter.</li> | + | <li><li> • Use barrier tips has filter.</li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>• Centrifuge</li> | + | <li><li> • Centrifuge</li> |
- | <li>• Liquid waste bin</li> | + | <li><li> • Liquid waste bin</li> |
- | <li>• 1,5 ml and 2 ml epps</li> | + | <li><li> • 1,5 ml and 2 ml epps</li> |
- | <li>• Plasmid Isolation Kit</li> | + | <li><li> • Plasmid Isolation Kit</li> |
- | <li>• Resuspension Buffer</li> | + | <li><li> • Resuspension Buffer</li> |
- | <li>• Lysis Buffer</li> | + | <li><li> • Lysis Buffer</li> |
- | <li>• Neutralization Buffer</li> | + | <li><li> • Neutralization Buffer</li> |
- | <li>• Wash Buffer </li> | + | <li><li> • Wash Buffer </li> |
- | <li>• Elution Buffer </li> | + | <li><li> • Elution Buffer </li> |
- | <li>• Vortex</li> | + | <li><li> • Vortex</li> |
- | <li>• 50-500 µl pipettes</li> | + | <li><li> • 50-500 µl pipettes</li> |
- | <li>• Timer</li> | + | <li><li> • Timer</li> |
- | <li>• Filter columns and epps</li> | + | <li><li> • Filter columns and epps</li> |
- | <li>• PCR Tubes (mini epp)</li> | + | <li><li> • PCR Tubes (mini epp)</li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>• Use gloves!</li> | + | <li><li> • Use gloves!</li> |
- | <li>• Liquid cultures centrifuge: 5 ml; 4100 rpm, 15 min. 1,5 ml; 13000 rpm, 10 min. (with 2 µl epp) </li> | + | <li><li> • Liquid cultures centrifuge: 5 ml; 4100 rpm, 15 min. 1,5 ml; 13000 rpm, 10 min. (with 2 µl epp) </li> |
- | <li>• After the centrifuge, the supernatent should be disposed without taking any pellets along with it.</li> | + | <li><li> • After the centrifuge, the supernatent should be disposed without taking any pellets along with it.</li> |
- | <li>• Add 250 µl Resuspension Solution. Vortex until to be homogeneous(about 30 sec.). You can pipetting too.</li> | + | <li><li> • Add 250 µl Resuspension Solution. Vortex until to be homogeneous(about 30 sec.). You can pipetting too.</li> |
- | <li>• Add 250 µl Lysis Solution. You must invert(4-6 times).</li> | + | <li><li> • Add 250 µl Lysis Solution. You must invert(4-6 times).</li> |
- | <li>• There must be 3 min.</li> | + | <li><li> • There must be 3 min.</li> |
- | <li>• Add 350 µl Neutralization Solution and invert(4-6 times).</li> | + | <li><li> • Add 350 µl Neutralization Solution and invert(4-6 times).</li> |
- | <li>• 5 min. centrifuge.</li> | + | <li><li> • 5 min. centrifuge.</li> |
- | <li>• After centrifuge try to take supernatent without pellets as much as you can to fitler columns.</li> | + | <li><li> • After centrifuge try to take supernatent without pellets as much as you can to fitler columns.</li> |
- | <li>• 1 min. (13 000 rpm) centrifuge. Throw the supernatent to liquid waste bin.</li> | + | <li><li> • 1 min. (13 000 rpm) centrifuge. Throw the supernatent to liquid waste bin.</li> |
- | <li>• Add 500 µl Wash Solution. 1 min. centrifuge. Throw the supernatent to liquid waste bin.</li> | + | <li><li> • Add 500 µl Wash Solution. 1 min. centrifuge. Throw the supernatent to liquid waste bin.</li> |
- | <li>• Add 500 µl Wash Solution. 1 min. centrifuge. Throw the supernatent to liquid waste bin.</li> | + | <li><li> • Add 500 µl Wash Solution. 1 min. centrifuge. Throw the supernatent to liquid waste bin.</li> |
- | <li>• 1 min. centrifuge to get rid of alcohol.</li> | + | <li><li> • 1 min. centrifuge to get rid of alcohol.</li> |
- | <li>• Throw the supernatent with epp.</li> | + | <li><li> • Throw the supernatent with epp.</li> |
- | <li>• Put new 1,5 epp under the filter column.</li> | + | <li><li> • Put new 1,5 epp under the filter column.</li> |
- | <li>• Transfer the columns into 1.5 ml microcentrifuge tubes and leave their caps open for 2 minutes so that the ethanol that they contain dissolves in air.</li> | + | <li><li> • Transfer the columns into 1.5 ml microcentrifuge tubes and leave their caps open for 2 minutes so that the ethanol that they contain dissolves in air.</li> |
- | <li>• Add 50 µl Elution Buffer.</li> | + | <li><li> • Add 50 µl Elution Buffer.</li> |
- | <li>• Incubate for 2 minutes.</li> | + | <li><li> • Incubate for 2 minutes.</li> |
- | <li>• Centrifuge for 2 minutes.</li> | + | <li><li> • Centrifuge for 2 minutes.</li> |
- | <li>• Take the supernatent to new epp.</li> | + | <li><li> • Take the supernatent to new epp.</li> |
- | <li>• Measure them with Nanodrop. </li> | + | <li><li> • Measure them with Nanodrop. </li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>• All centrifuges are 13000 rpm.</li> | + | <li><li> • All centrifuges are 13000 rpm.</li> |
- | <li>• Don’t shake Lysis Solution.</li> | + | <li><li> • Don’t shake Lysis Solution.</li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>•LB-Agar(chemical storage)</li> | + | <li><li> •LB-Agar(chemical storage)</li> |
- | <li>•Sterile dH2O</li> | + | <li><li> •Sterile dH2O</li> |
- | <li>•Flask </li> | + | <li><li> •Flask </li> |
- | <li>•Liquid autoclave</li> | + | <li><li> •Liquid autoclave</li> |
- | <li>•Aluminium folio</li> | + | <li><li> •Aluminium folio</li> |
- | <li>•Antibiotic</li> | + | <li><li> •Antibiotic</li> |
- | <li>•Empty plates</li> | + | <li><li> •Empty plates</li> |
- | <li>•Parafilm</li> | + | <li><li> •Parafilm</li> |
</ul> | </ul> | ||
</div> | </div> | ||
Line 1,219: | Line 2,335: | ||
<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>•In a steril environment, the tare of the container should be measured and subtracted from the overall weight.</li> | + | <li><li> •In a steril environment, the tare of the container should be measured and subtracted from the overall weight.</li> |
- | <li>•7 grams of LB Agar is put in the container.(If there is no LB agar, add the 4 gram LB broth and 2,7 gram agar.)</li> | + | <li><li> •7 grams of LB Agar is put in the container.(If there is no LB agar, add the 4 gram LB broth and 2,7 gram agar.)</li> |
- | <li>•Add 200 ml distilled water to graduated cyclindar.</li> | + | <li><li> •Add 200 ml distilled water to graduated cyclindar.</li> |
- | <li>•These two are mixed in a beaker.</li> | + | <li><li> •These two are mixed in a beaker.</li> |
- | <li>•When you are opening the beaker be sure that it doesn’t contact with air.</li> | + | <li><li> •When you are opening the beaker be sure that it doesn’t contact with air.</li> |
- | <li>•Autoclave tape is sticked on to the aliminium.</li> | + | <li><li> •Autoclave tape is sticked on to the aliminium.</li> |
- | <li>•The beaker is placed in to the autoclave machine.</li> | + | <li><li> •The beaker is placed in to the autoclave machine.</li> |
- | <li>•Autoclave machine has to have distilled water or demineralized water. The water level in the autoclave machine has to be a little higher than the liquid level of the beaker.</li> | + | <li><li> •Autoclave machine has to have distilled water or demineralized water. The water level in the autoclave machine has to be a little higher than the liquid level of the beaker.</li> |
- | <li>•Then start the 90 min.</li> | + | <li><li> •Then start the 90 min.</li> |
- | <li>•Take out the beaker and add antibiotics if required.</li> | + | <li><li> •Take out the beaker and add antibiotics if required.</li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>•Don’t be far away fire.</li> | + | <li><li> •Don’t be far away fire.</li> |
- | <li>•Don’ forget to write the date.</li> | + | <li><li> •Don’ forget to write the date.</li> |
- | <li>•Don't forget add the amtibiotics</li> | + | <li><li> •Don't forget add the amtibiotics</li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>•LB-Broth(chemical storage)</li> | + | <li><li> •LB-Broth(chemical storage)</li> |
- | <li>•Sterile dH2O</li> | + | <li><li> •Sterile dH2O</li> |
- | <li>•Flask </li> | + | <li><li> •Flask </li> |
- | <li>•Lab skale</li> | + | <li><li> •Lab skale</li> |
- | <li>•Liquid autoclave</li> | + | <li><li> •Liquid autoclave</li> |
- | <li>•Alumininum folio</li> | + | <li><li> •Alumininum folio</li> |
- | <li>•Antibiotic</li> | + | <li><li> •Antibiotic</li> |
</ul> | </ul> | ||
</div> | </div> | ||
Line 1,274: | Line 2,390: | ||
<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>•In a steril environment, the tare of the container should be measured and subtracted from the overall weight.</li> | + | <li><li> •In a steril environment, the tare of the container should be measured and subtracted from the overall weight.</li> |
- | <li>•4 grams of LB Broth is put in the container.</li> | + | <li><li> •4 grams of LB Broth is put in the container.</li> |
- | <li>•Add 200 ml distilled water to graduated cyclindar.</li> | + | <li><li> •Add 200 ml distilled water to graduated cyclindar.</li> |
- | <li>•These two are mixed in a beaker.</li> | + | <li><li> •These two are mixed in a beaker.</li> |
- | <li>•When you are opening the beaker be sure that it doesn’t contact with air.</li> | + | <li><li> •When you are opening the beaker be sure that it doesn’t contact with air.</li> |
- | <li>•Autoclave tape is sticked on to the aliminium.</li> | + | <li><li> •Autoclave tape is sticked on to the aliminium.</li> |
- | <li>•The beaker is placed in to the autoclave machine.</li> | + | <li><li> •The beaker is placed in to the autoclave machine.</li> |
- | <li>•Autoclave machine has to have distilled water or demineralized water. The water level in the autoclave machine has to be a little higher than the liquid level of the beaker.</li> | + | <li><li> •Autoclave machine has to have distilled water or demineralized water. The water level in the autoclave machine has to be a little higher than the liquid level of the beaker.</li> |
- | <li>•Then start the 90 min.</li> | + | <li><li> •Then start the 90 min.</li> |
- | <li>•Take out the beaker and add antibiotics if required.</li> | + | <li><li> •Take out the beaker and add antibiotics if required.</li> |
</ul> | </ul> | ||
</div> | </div> | ||
Line 1,289: | Line 2,405: | ||
<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>• Don’t be far away fire.</li> | + | <li><li> • Don’t be far away fire.</li> |
- | <li>• Don’ forget to write date </li> | + | <li><li> • Don’ forget to write date </li> |
</ul> | </ul> | ||
</div> | </div> | ||
Line 1,316: | Line 2,432: | ||
<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>• PCR Tube</li> | + | <li><li> • PCR Tube</li> |
- | <li>• Plasmids</li> | + | <li><li> • Plasmids</li> |
- | <li>• 10× T4 DNA Ligase Buffer</li> | + | <li><li> • 10× T4 DNA Ligase Buffer</li> |
- | <li>• T4 DNA Ligase</li> | + | <li><li> • T4 DNA Ligase</li> |
- | <li>• Nuklease Free H2O</li> | + | <li><li> • Nuklease Free H2O</li> |
- | <li>• Heat Block</li> | + | <li><li> • Heat Block</li> |
</ul> | </ul> | ||
</div> | </div> | ||
Line 1,327: | Line 2,443: | ||
<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>• Take PCR Tube ® Write Lig on it</li> | + | <li><li> • Take PCR Tube ® Write Lig on it</li> |
- | <li>• Add 2µl upstream, 2µl downstream, 2µl destination plasmids</li> | + | <li><li> • Add 2µl upstream, 2µl downstream, 2µl destination plasmids</li> |
- | <li>• Add 2µl T4 DNA Ligase buffer</li> | + | <li><li> • Add 2µl T4 DNA Ligase buffer</li> |
- | <li>• Add 1µl T4 DNA Ligase</li> | + | <li><li> • Add 1µl T4 DNA Ligase</li> |
- | <li>• Add 11µl nüklease free H2O. It must be 20 µl.</li> | + | <li><li> • Add 11µl nüklease free H2O. It must be 20 µl.</li> |
- | <li>• It must wait 37 C (10 min.), 80⁰C(20 min.)</li> | + | <li><li> • It must wait 37 C (10 min.), 80⁰C(20 min.)</li> |
- | <li>• After incubation you must followwith transformation. The only difference between normal transformation and lig’s transformation is adding 2µl plasmid.</li> | + | <li><li> • After incubation you must followwith transformation. The only difference between normal transformation and lig’s transformation is adding 2µl plasmid.</li> |
</ul> | </ul> | ||
</div> | </div> | ||
Line 1,339: | Line 2,455: | ||
<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>• You must leave plasmid in -20°C after lig.</li> | + | <li><li> • You must leave plasmid in -20°C after lig.</li> |
- | <li>• If electrophoresis results are successful you will follow with lig.</li> | + | <li><li> • If electrophoresis results are successful you will follow with lig.</li> |
</ul> | </ul> | ||
</div> | </div> | ||
Line 1,366: | Line 2,482: | ||
<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>• Falkon</li> | + | <li><li> • Falkon</li> |
- | <li>• Pipette gun</li> | + | <li><li> • Pipette gun</li> |
- | <li>• 5-10 ml pipette</li> | + | <li><li> • 5-10 ml pipette</li> |
- | <li>• Ice box</li> | + | <li><li> • Ice box</li> |
- | <li>• LB-Broth</li> | + | <li><li> • LB-Broth</li> |
- | <li>• Antibiotic</li> | + | <li><li> • Antibiotic</li> |
- | <li>• Colony</li> | + | <li><li> • Colony</li> |
- | <li>• Pipette</li> | + | <li><li> • Pipette</li> |
- | <li>• Shaker</li> | + | <li><li> • Shaker</li> |
- | <li>• Incubator</li> | + | <li><li> • Incubator</li> |
</ul> | </ul> | ||
</div> | </div> | ||
Line 1,382: | Line 2,498: | ||
<ul > | <ul > | ||
<li>In sterile environment,</li> | <li>In sterile environment,</li> | ||
- | <li>• Take falkons.</li> | + | <li><li> • Take falkons.</li> |
- | <li>• Add 5 ml LB-Broth into falkons with pipette gun.</li> | + | <li><li> • Add 5 ml LB-Broth into falkons with pipette gun.</li> |
- | <li>• Take an antibiotic into ice.</li> | + | <li><li> • Take an antibiotic into ice.</li> |
- | <li>• Add antibiotic.</li> | + | <li><li> • Add antibiotic.</li> |
- | <li>• Take the colony from plate.</li> | + | <li><li> • Take the colony from plate.</li> |
- | <li>• Leave it into falkon.</li> | + | <li><li> • Leave it into falkon.</li> |
- | <li>• Take falkons to the shaker.(it can be 240 or 320 rpm) Leave it 37⁰C ( 12-14 hours ) incubator.</li> | + | <li><li> • Take falkons to the shaker.(it can be 240 or 320 rpm) Leave it 37⁰C ( 12-14 hours ) incubator.</li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>• Open the program(Nano- Spech).</li> | + | <li><li> • Open the program(Nano- Spech).</li> |
- | <li>• Choose dsDNA.</li> | + | <li><li> • Choose dsDNA.</li> |
- | <li>• Be sure pipette is 2 µl.</li> | + | <li><li> • Be sure pipette is 2 µl.</li> |
- | <li>• Leave the drop middle.</li> | + | <li><li> • Leave the drop middle.</li> |
- | <li>• First try Elution Buffer (for blanck)</li> | + | <li><li> • First try Elution Buffer (for blanck)</li> |
- | <li>• Click blank.</li> | + | <li><li> • Click blank.</li> |
- | <li>• If results are about 0(zero) its okay.</li> | + | <li><li> • If results are about 0(zero) its okay.</li> |
- | <li>• Write their names, click "start".</li> | + | <li><li> • Write their names, click "start".</li> |
- | <li>• Nucleic Acid must be much over than 17 ng.</li> | + | <li><li> • Nucleic Acid must be much over than 17 ng.</li> |
<li> You must check 2 times and to take their avarage. </li> | <li> You must check 2 times and to take their avarage. </li> | ||
</ul> | </ul> | ||
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<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>• 1.5 mL epps</li> | + | <li><li> • 1.5 mL epps</li> |
- | <li>• Heat Block (42ºC)</li> | + | <li><li> • Heat Block (42ºC)</li> |
- | <li>• Competent cells</li> | + | <li><li> • Competent cells</li> |
- | <li>• LB-Broth</li> | + | <li><li> • LB-Broth</li> |
- | <li>• Ice</li> | + | <li><li> • Ice</li> |
- | <li>• Ice Box</li> | + | <li><li> • Ice Box</li> |
- | <li>• Microcentrifuge</li> | + | <li><li> • Microcentrifuge</li> |
- | <li>• Rocks for epp</li> | + | <li><li> • Rocks for epp</li> |
- | <li>• Timer</li> | + | <li><li> • Timer</li> |
- | <li>• LB agar plates</li> | + | <li><li> • LB agar plates</li> |
- | <li>• Spreader</li> | + | <li><li> • Spreader</li> |
- | <li>• Shaker (240 rpm)</li> | + | <li><li> • Shaker (240 rpm)</li> |
- | <li>• Parafilm</li> | + | <li><li> • Parafilm</li> |
- | <li>• Band</li> | + | <li><li> • Band</li> |
- | <li>• 1 µl pipette (for plasmid)</li> | + | <li><li> • 1 µl pipette (for plasmid)</li> |
- | <li>• 50µl pipette</li> | + | <li><li> • 50µl pipette</li> |
- | <li>• 150 µl pipette (for spread)</li> | + | <li><li> • 150 µl pipette (for spread)</li> |
- | <li>• 450 and 500 µl pipettes (for LB)</li> | + | <li><li> • 450 and 500 µl pipettes (for LB)</li> |
- | <li>• Incubator (37 C)</li> | + | <li><li> • Incubator (37 C)</li> |
- | <li>• Alcohol</li> | + | <li><li> • Alcohol</li> |
- | <li>• If there is diluting from the kit:</li> | + | <li><li> • If there is diluting from the kit:</li> |
- | <li>• Nuclease Free H2O</li> | + | <li><li> • Nuclease Free H2O</li> |
- | <li>• 15 µl pipette</li> | + | <li><li> • 15 µl pipette</li> |
- | <li>• PCR Tube (Mini epp)</li> | + | <li><li> • PCR Tube (Mini epp)</li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>• Sterilize the environment.</li> | + | <li><li> • Sterilize the environment.</li> |
- | <li>• Take the competent cells from -80°C.</li> | + | <li><li> • Take the competent cells from -80°C.</li> |
- | <li>• Turn on the heat block (42⁰C)</li> | + | <li><li> • Turn on the heat block (42⁰C)</li> |
- | <li>• Put 1 µl plasmid in a 1,5 ml ep.</li> | + | <li><li> • Put 1 µl plasmid in a 1,5 ml ep.</li> |
- | <li>• Put competent cells in the same 1,5 ml epp. Use 50µl competent cells.</li> | + | <li><li> • Put competent cells in the same 1,5 ml epp. Use 50µl competent cells.</li> |
- | <li>• Place the epps into the microcentrifuge. (30-60 sec., 3000 rpm)</li> | + | <li><li> • Place the epps into the microcentrifuge. (30-60 sec., 3000 rpm)</li> |
- | <li>• Incubate on ice for 45 min. </li> | + | <li><li> • Incubate on ice for 45 min. </li> |
- | <li>• Put epps into the heat block at 42⁰C for 120 sec.</li> | + | <li><li> • Put epps into the heat block at 42⁰C for 120 sec.</li> |
- | <li>• Put epps back on ice for 5 min. to reduce damage to the e.coli cells.</li> | + | <li><li> • Put epps back on ice for 5 min. to reduce damage to the e.coli cells.</li> |
- | <li>• Add 450 ul of LB(with no antibiotic added)</li> | + | <li><li> • Add 450 ul of LB(with no antibiotic added)</li> |
- | <li>• Incubate examples on shaker for 1 hour at 37⁰C. (240 rpm)</li> | + | <li><li> • Incubate examples on shaker for 1 hour at 37⁰C. (240 rpm)</li> |
- | <li>• Spread about 135 ul of the final examples on LB plates (with appropriate antibiotic added-usually Ampicillin or Kanamycin) </li> | + | <li><li> • Spread about 135 ul of the final examples on LB plates (with appropriate antibiotic added-usually Ampicillin or Kanamycin) </li> |
- | <li>• Incubate overnight.</li> | + | <li><li> • Incubate overnight.</li> |
- | <li>• Pick colonies about 14-16 hours later</li> | + | <li><li> • Pick colonies about 14-16 hours later</li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="itemlist"> | <div class="itemlist"> | ||
<ul > | <ul > | ||
- | <li>• Don’t use gloves when you are working with fire. | + | <li><li> • Don’t use gloves when you are working with fire. |
- | <li>• Don’t forget to write notes on epps. | + | <li><li> • Don’t forget to write notes on epps. |
- | <li>• Be quick while sirkini Wilhelm compatent cells | + | <li><li> • Be quick while sirkini Wilhelm compatent cells |
</ul> | </ul> | ||
</div> | </div> | ||
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</div> | </div> | ||
</div> | </div> | ||
+ | |||
+ | |||
<div class="col1"> | <div class="col1"> | ||
<div class="col4 magRight2"> | <div class="col4 magRight2"> | ||
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</div> | </div> | ||
</div> | </div> | ||
+ | |||
+ | |||
<div class="col1"> | <div class="col1"> | ||
<div class="col4 magRight2"> | <div class="col4 magRight2"> | ||
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<p><i style="color:white">...Sentegen</i> for helping us to to provide our genes, special thanks for cool centrifuge.</p> | <p><i style="color:white">...Sentegen</i> for helping us to to provide our genes, special thanks for cool centrifuge.</p> | ||
<p>...the whole team, especially to our advisors: <i style="color:white"> Fatma Betül Çevik, Ayşe Demirci, Esma Ölmez </i> who were always with us.</p> | <p>...the whole team, especially to our advisors: <i style="color:white"> Fatma Betül Çevik, Ayşe Demirci, Esma Ölmez </i> who were always with us.</p> | ||
+ | <p>Also we want to thank to <i>Muradiye Acar, Burak Yılmaz, Fazilet Yılmaz, Gökhan Nas, Ömer Faruk Hatipoğlu</i> for their helps about our Project subject also our experiments. | ||
+ | And thanks to <i> genetic students </i> for their helps about our experiments.</p> | ||
<p style="color:white"><b>Wiki</b></p> | <p style="color:white"><b>Wiki</b></p> | ||
- | <p><i style="color:white">.. | + | <p><i style="color:white">... Havva Gulay Gurbuz</i> – graduate student at Bilkent University. She spent a lot of her time for us. </p> |
<p><i style="color:white">... Busra Nur Aydın</i> who helped us with designing - she has magical hands.:)</p> | <p><i style="color:white">... Busra Nur Aydın</i> who helped us with designing - she has magical hands.:)</p> | ||
- | <p><i style="color:white">. | + | <p><i style="color:white">.. Kasım Özkan </i> who helped us with doing our comics.</p> |
<p style="color:white"><b>Funding</b></p> | <p style="color:white"><b>Funding</b></p> | ||
- | <p>We want to thank <i style="color:white"> | + | <p>We want to thank <i style="color:white"> Osman Birgin </i>, who is the head of Energy Market Regulatory Authory for finding us generous funds. He did things which mean a lot for us.</p> |
<p>We want to thank <i style="color:white"> our school </i> again in this chapter. They provided their sources for us. </p> | <p>We want to thank <i style="color:white"> our school </i> again in this chapter. They provided their sources for us. </p> | ||
<p>Beyond these, we want to thank <i style="color:white"> Turgut Ozal 2013 Collegiate iGEMers </i> for being our advisors. We also want to give a special thank to <i style="color:white"> Esin and Kübra Sisters </i> for yummy meals :)</p> | <p>Beyond these, we want to thank <i style="color:white"> Turgut Ozal 2013 Collegiate iGEMers </i> for being our advisors. We also want to give a special thank to <i style="color:white"> Esin and Kübra Sisters </i> for yummy meals :)</p> | ||
- | <p>There is also one more group that we want to thank. They were supporting us in our desperative times. <i style="color:white"> Our parents! </i> Especially to Beril’s mom <i style="color:white"> | + | <p>There is also one more group that we want to thank. They were supporting us in our desperative times. <i style="color:white"> Our parents! </i> Especially to Beril’s mom <i style="color:white"> Hülya Gürdap </i> for baklava (Turkish dessert) and to Dilara’s mom <i style="color:white"> Özlem Soylu </i> for transportation.</p> |
- | <p>Aaaand finally: Our instructor <i style="color:white"> | + | <p>Aaaand finally: Our instructor <i style="color:white"> Mihriban Ozgun </i> for her amazing "instructorhood" </p> |
</div> | </div> | ||
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</li> | </li> | ||
- | + | <li id="pageContact"> | |
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+ | <h2>Contact Us</h2> | ||
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+ | <a href="mailto:ngss.aei.turkey@gmail.com"><p class="padBot4">Email: ngss.aei.turkey@gmail.com </p></a> | ||
+ | <a href="http://twitter.com/NGSS_AEI_TURKEY"><p class="padBot4">Our Twitter Page : NGSS_AEI_TURKEY</p></a> | ||
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</ul> | </ul> | ||
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<div class="mainFooter"> | <div class="mainFooter"> | ||
<div class="privHold"> | <div class="privHold"> | ||
- | < | + | <img src="https://static.igem.org/mediawiki/igem.org/1/16/Sponsors.png" width="500px" /> |
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<td valign="middle"><a href="http://sentegen.com/" target="_blank"><img src="https://static.igem.org/mediawiki/2013hs/a/a7/Sentegen.png" width="200"/></a></td> | <td valign="middle"><a href="http://sentegen.com/" target="_blank"><img src="https://static.igem.org/mediawiki/2013hs/a/a7/Sentegen.png" width="200"/></a></td> | ||
- | <td valign="middle"><a href="http://en.turgutozal.edu.tr/" target="_blank"><img src="https://static.igem.org/mediawiki/2013hs/7/74/Turgutozal.png" width=" | + | |
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Latest revision as of 05:11, 22 June 2013