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"

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.

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.

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.

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

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

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

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

The LasI's connects with LasR molecules in receiver cells and activates pLas.

LacI influence represses by adding IPTG to the medium and in our sender cells LasI produce starts and spreads.

 

 

 

Also Endolysin drilss outer layer. Therefore the bacteria goes through die.

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.

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

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.

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.

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.

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

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!

https://twitter.com/SubBacillus

https://twitter.com/E_coli1885

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

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.

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

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.

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

Click here for our powerpoint slide in Turkish

Click here for our powerpoint slide in English

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

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

06.01.2013 Saturday

06.02.2013 Sunday

06.03.2013 Monday

06.04.2013 Tuesday

06.05.2013 Wednesday

06.06.2013 Thursday

06.07.2013 Friday

06.08.2013 Saturday

06.09.2013 Sunday

06.10.2013 Monday

06.11.2013 Tuesday

06.12.2013 Wednesday

06.13.2013 Thursday

06.14.13 Friday

06.15.13 Saturday

06.16.13 Sunday

06.17.13 Monday

18.06.13 Tuesday

06.19.13 Wednesday

06.20.13 Thursday

06.21.13 Friday

Hey Guys,

This is our Comics. Hope oyu like it. Next year it will contain more and more colour

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

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!"

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Safety is an important issue for lab experiments. We have created a safety text and also safety video! The name of our video is “Lab over Clouds”. You can find our safety text just under the video.

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"

Email: ngss.aei.turkey@gmail.com

Our Twitter Page : NGSS_AEI_TURKEY