Team:Beijing HDFLS High/Project

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== TITLE: The UV-responsive Wintergreen Odor Generator==
== ABSTRACT ==
== ABSTRACT ==
The UV-responsive wintergreen odor generator catalyzes the conversion of the precursor salicylic acid to the wintergreen odor methyl salicylate under extensive UV irradiation. The biosynthetic device is composed of two transcriptional devices: Rec A (SOS) promoter (BBa_J22106) and a wintergreen odor enzyme generator (BBa_J45119). The odor generator takes as input extensive UV irradiation and produce as output an enzyme that catalyzes production of an odor from a chemical precursor.
The UV-responsive wintergreen odor generator catalyzes the conversion of the precursor salicylic acid to the wintergreen odor methyl salicylate under extensive UV irradiation. The biosynthetic device is composed of two transcriptional devices: Rec A (SOS) promoter (BBa_J22106) and a wintergreen odor enzyme generator (BBa_J45119). The odor generator takes as input extensive UV irradiation and produce as output an enzyme that catalyzes production of an odor from a chemical precursor.
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[[Image:Generator.JPG|center|600px]]
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[[Image:Generator.JPG|center|800px]]
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===INTRODUCTION===
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We designed a biological system using ''E.coli'' to detect extensive UV irradiation. If the toolkit is under extensive UV irradiation, it will produce a wintergreen odor.Then, This special odor will warn people of the UV irradiation. In order to increase the attraction of our product, we make ''E.coli'' culture medium with the different color using red, yellow and blue pigments.<br><br>
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[[Image:Outlook.jpg||Center||400px|]]<Br>
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<br>
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<br>
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Ultraviolet (UV) light is part of the electromagnetic spectrum having waves with frequencies higher than those that humans can identify as the color violet. These frequencies are invisible to humans, but visible to a number of insects and birds.<br>
 +
 +
UVC
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The shortest one of UV rays ，usually do not penetrate the Earth’s ozone layer.
 +
<br>
 +
UVB
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Longer waves that reach skin’s surface – can cause surface tanning burning ,and  signs of aging.
 +
<br>
 +
UVA
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Even longer waves that can penetrate deep into the skin’s surface ,releasing free radicals and causing DNA changes that can result in skin cancers.<br>
 +
 
 +
[[File:uv.jpg||center||800px|]]
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<br>Recently, because the ozone layer has been destroying by some artificial chemical substances like CFC (Freon),the increase of UV irradiation appears on the earth.
 +
[[Image:Beijing_HDFLS_High_ESA.jpg||center|||800px|]]
 +
<br>
 +
Extensive UV irradiation is harmful to human being, such as
 +
* 1) Weakening of the immune system
 +
* 2) Increased risk of skin cancer, cataracts
 +
* 3) Causing heavy damage to genes
== PARTS==
== PARTS==
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[[Image:PARTS.JPG|center|600px]]
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[[Image:1.jpg|center|700px]]
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<br>
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Extensive UV irradiation impings on heavily DNA damage and results in a single-strand DNA occuring, which activates RecA protein.Then,RecA protein leads to the expression of about 15 different repair proteins to rescue DNA damage.RecA protein is the central regulator in SOS repair system.Rec A (SOS) Promoter is available in 2004 registry part plate.
 +
[[Image:3.jpg|center|700px]]
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BBa_J45119 takes as input a transcriptional signal (PoPS) and produce as output the BMST1 enzyme that catalyzes production of methyl salicylate from salicylic acid. Methyl salicylate has a wintergreen smell. Wintergreen odor enzyme (BMST1) generator is availabe in 2005 registry part plate.
 +
[[Image:4.jpg|center|700px]]
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== Parts ==
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== RESULTS ==
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=== Promoter :RecA(BBa_J22106) ===
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=== Growing Part A and Part B ===
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e chose to start with the promoter RecA , RecA is a recombinase enzyme, i.e. a DNA strand exchange and recombination protein with protease and nuclease activity. The RecA protein binds strongly and in long clusters to ssDNA to form a nucleoprotein filament. The protein has more than one DNA binding site, and thus can hold a single strand and double strand together. This feature makes it possible to catalyze a DNA synapsis reaction between a DNA double helix and a complementary region of single stranded DNA. And ReaA is the standard gene for the SOS-response therefore it is well characterized. Other scientist have been successful when using recA for gene regulation.
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After Growing Part A &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;After Growing Part B
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=== SOS RESPONSE ===
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[[Image:浩泽菌落1.jpg|left|350px]]
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The SOS response was discovered and named by Miroslav Radman in 1975.The SOS response is a global response to DNA damage in which the cell cycle is arrested and DNA repair and mutagenesis are induced. The SOS system is the repair system of the cell to counteract extensive DNA damages e.g. caused by UV radiation or ionizing radiation. If UV radiation impinges on the bacterial DNA it may cause cross-linking of adjacent cytosine and thymine bases resulting in pyrimidine dimers – a process called direct DNA damage. Heavily damaged DNA is no longer able to replicate and leads to interruption of the cell cycle. The SOS response allows bacterial cells to repair damaged DNA and continue the cell cycle. In normal cells, the SOS system is turned off and it is only activated in the case of DNA damage. The central regulator of the SOS response is RecA. The RecA protein is activated by single-stranded DNA occurring during heavy DNA damage. Activated RecA leads to the cleavage of the LexA-repressor protein that normally blocks the RNA-polymerase for transcription of repair proteins. The inactivated LexA can no longer bind to the DNA strain which allows for the production of a cascade of about 20 different repair proteins, amongst others SulA, UvrB and DinF. Another action of the active LexA repressor protein is regulation of its own transcription as well as transcription of RecA . When DNA repair is done and the activity of precA has decreased the system returns to its steady state.
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[[Image:浩泽菌落2.jpg|right|350px]]
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<br>
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<br>
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<br><br>
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<br>
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<br><br>
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<br>
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<br><br>
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<br>
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<br><br>
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<br>
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<br><br><br>
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<br>
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=== REPORTER : "Wintergreen odor enzyme (BMST1) generator" (BBa_J45119) ===
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=== Resriction digest of Part A and Part B ===
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[[Image:beijing_HDFLS_project_odor.jpg|center|500px]]
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[[Image:Digestion.jpg|center|500px]]
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The wintergreen odor generator (BBa_J45120) catalyzes the conversion of the precursor salicylic acid to the odor methyl salicylate that has a wintergreen smell. The biosynthetic device is composed of two transcriptional devices: a constitutive transcription source (BBa_R0040) and an odor enzyme generator (BBa_J45119). Odor enzyme generators take as input a transcriptional signal and produce as output an enzyme that catalyzes production of an odor from a chemical precursor.
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== Method ==
 
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=== Experimental theory ===
 
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“3A(antibiotic)” Assembly is one way of assembling two strandard biological parts together. “3A(antibiotic)” Assembly uses the restriction sites  in the prefix and suffix of the two parts to assemble them into the new composite part. The composite part will have the same prefix and suffix as the ‘parent’ parts.
 
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[[Image:beijing_HDFLS_project_schematic.JPG|center|500px]]
 
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This time choose RecA as promoter and wintergreen odor biosynthetic system as reporter. The uesful part of RecA are cut out by  enzymese ECORI and PSTI ,and we also need ECORI and PSTI to cut the useful part of wintergreen odor biosynthetic system ,and then we need to connect them together to completely the whole system.
 
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== Step ==
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[[Image:PARTAandB.JPG|center|500px]]
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=== STEP1: GROWING ===
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=== Growing After Ligation ===
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[[Image:ligation.jpg|center|310px]]
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<br>
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[[Image:lala.jpg|center|310px]]
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=== Restriction digest after ligation ===
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[[Image:AB.png|center|800px]]
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[[Image:PARTAB.JPG|center|500px]]
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=== DNA Sequencing and Submitting Part===
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http://parts.igem.org/Part:BBa_K994000
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[[Image:Bases.jpg|center|800px]]
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== STEP==
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=== STEP1: TRANSFORMATION AND GROWING ===
(Make sure we have enough copies of BBa_J22106 and BBa_J45119.)
(Make sure we have enough copies of BBa_J22106 and BBa_J45119.)
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*PART1:
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[[Image:Growing.jpg|center|600px]]
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**Clean the lab bench
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=== STEP2: Plamid MINIPREP ===
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**Label the agar plates with names of partA and parB and partC (partA:J22106  partB:J45119 partC: BBa_J45199banana)
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[[Image:PLASMID.jpg|center|600px]]
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**Use an inoculating loop to transfer some cells from the PartA agar stab to the appropriate agar plate .Dip an inoculating loop into the stab at the same location, and streak the bacteria onto the agar plate in a zig-zag pattern
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**Repeat step 3 for partB and partC
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=== STEP3：RESTRICTION DIGEST ===
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**Place the agar plates into the incubator with the agar facing down. Incubate the agar plates at 37 degrees for 14-16hours. Alternately, incubate at room temperature for 24-30 hours.
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[[Image:Qiyue.JPG|center|600px]]
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*PART2
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<br>
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**Clean the lab bench
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**Remove the agar plates from fridge
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[[Image:20130518 102210.jpg|center|600px]]
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**Add 5ml of LB broth to each culture tube(with 14ml )
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**Use an inoculating loop to pick a single colony from each agar plate and sent it into the culture tube.
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**Incubate for 16 hours at 37 degrees in a shaker or rotator.
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**Seal until use
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=== STEP2:MINIPREP ===
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=== STEP3：RESTRICTION DIGEST ===  
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=== STEP4：LIGATION ===
=== STEP4：LIGATION ===
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[[Image:Menxuyang.jpg|center|600px]]
=== STEP5：TRANSFORMATION ===
=== STEP5：TRANSFORMATION ===
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=== STEP6：MEASUREMENT AND COMPARE ===  
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=== STEP7：DESGIN AND COMPLETE ===
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=== STEP6：MINIPREP ===
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=== STEP7：RESTRICTION DIGEST ===
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=== STEP8：DNA SEQUENCING===
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=== STEP9：UV INDUCTION===
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=== STEP10：DESGIN AND COMPLETE ===
 +
[[Image:Progect3.jpg|center|600px]]

Latest revision as of 03:42, 22 June 2013

Contents

TITLE: The UV-responsive Wintergreen Odor Generator

ABSTRACT

The UV-responsive wintergreen odor generator catalyzes the conversion of the precursor salicylic acid to the wintergreen odor methyl salicylate under extensive UV irradiation. The biosynthetic device is composed of two transcriptional devices: Rec A (SOS) promoter (BBa_J22106) and a wintergreen odor enzyme generator (BBa_J45119). The odor generator takes as input extensive UV irradiation and produce as output an enzyme that catalyzes production of an odor from a chemical precursor.

Generator.JPG

INTRODUCTION

We designed a biological system using E.coli to detect extensive UV irradiation. If the toolkit is under extensive UV irradiation, it will produce a wintergreen odor.Then, This special odor will warn people of the UV irradiation. In order to increase the attraction of our product, we make E.coli culture medium with the different color using red, yellow and blue pigments.

Outlook.jpg


Ultraviolet (UV) light is part of the electromagnetic spectrum having waves with frequencies higher than those that humans can identify as the color violet. These frequencies are invisible to humans, but visible to a number of insects and birds.

UVC The shortest one of UV rays ，usually do not penetrate the Earth’s ozone layer.
UVB Longer waves that reach skin’s surface – can cause surface tanning burning ,and signs of aging.
UVA Even longer waves that can penetrate deep into the skin’s surface ,releasing free radicals and causing DNA changes that can result in skin cancers.

Uv.jpg


Recently, because the ozone layer has been destroying by some artificial chemical substances like CFC (Freon),the increase of UV irradiation appears on the earth.

Beijing HDFLS High ESA.jpg


Extensive UV irradiation is harmful to human being, such as

  • 1) Weakening of the immune system
  • 2) Increased risk of skin cancer, cataracts
  • 3) Causing heavy damage to genes

PARTS

1.jpg


Extensive UV irradiation impings on heavily DNA damage and results in a single-strand DNA occuring, which activates RecA protein.Then,RecA protein leads to the expression of about 15 different repair proteins to rescue DNA damage.RecA protein is the central regulator in SOS repair system.Rec A (SOS) Promoter is available in 2004 registry part plate.

3.jpg

BBa_J45119 takes as input a transcriptional signal (PoPS) and produce as output the BMST1 enzyme that catalyzes production of methyl salicylate from salicylic acid. Methyl salicylate has a wintergreen smell. Wintergreen odor enzyme (BMST1) generator is availabe in 2005 registry part plate.

4.jpg

RESULTS

Growing Part A and Part B

After Growing Part A                                                                                                After Growing Part B

浩泽菌落1.jpg
浩泽菌落2.jpg



















Resriction digest of Part A and Part B

Digestion.jpg


PARTAandB.JPG

Growing After Ligation

Ligation.jpg


Lala.jpg

Restriction digest after ligation

AB.png


PARTAB.JPG

DNA Sequencing and Submitting Part

http://parts.igem.org/Part:BBa_K994000

Bases.jpg

STEP

STEP1: TRANSFORMATION AND GROWING

(Make sure we have enough copies of BBa_J22106 and BBa_J45119.)

Growing.jpg

STEP2: Plamid MINIPREP

600px

STEP3：RESTRICTION DIGEST

Qiyue.JPG


20130518 102210.jpg

STEP4：LIGATION

Menxuyang.jpg

STEP5：TRANSFORMATION

STEP6：MINIPREP

STEP7：RESTRICTION DIGEST

STEP8：DNA SEQUENCING

STEP9：UV INDUCTION

STEP10：DESGIN AND COMPLETE

Progect3.jpg
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