Team:Heidelberg LSL/Project intro

From 2012hs.igem.org

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<h2> Introduction </h2>
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Introduction:
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The basic idea of our project is to create a simple and cheap tool to measure UV-/ radioactive-mediated damage in living cells.
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We developed a biological system in E. coli which can react even to different quantities of UV-radiation. The grade of induced damage can be estimated by means of a colorful change of the bacterial suspension.
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This can be achieved by using the natural SOS response of E. coli. The SOS response is the universe Repair system of prokaryotic cells that response on DNA damage. These mechanisms are very efficient, because they act immediately after the DNA was damaged and repair precisely. This is important, because even small changes in the DNA can have serious consequences. Because of the efficiency the mechanisms can be a high sensitive detector of radiation for us. The the mechanism of the SOS response is further explained [[link|here]].
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We have made a construct using a synthetic plasmid backbone with an ORI-region and a selection marker and inserted one of the promoters of a repair protein, followed by the sequence of an enzyme for the colour reaction.
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Have have chosen to start with the promoter RecA and SulA. These promoters where the most suitable for our project. We have listed all advantages and disadvantages for us of both promoters:
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RecA:
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Recombines Enzym (DNA strand exchange and recombination protein with protease and nuclease activity)
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Advantages:
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• Standard gene for SOS-Response
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• researched and positive tested by other teams
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• easy to get in the partsregistry: http://partsregistry.org/wiki/index.php/Part:BBa_K154000
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• Graduation with recB, recC etc possible
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Disadvantages:
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• recA carries out several functions in the E.coli, thus it may be activated even if no radiation is present - might cause disturbance in our data
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SulA:
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SOS cell division inhibitor
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Advantages:
 +
• is only activated for the SOS-response - data would be correct
 +
• too short for partsregistry but the oligos can be easily designed and ordered by using the gene code from the partsregistry http://partsregistry.org/wiki/index.php/Part:BBa_K518010
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• used by other iGEM teams
 +
Disadvantages:
 +
not sure if graduations sulB, sulC etc will work for us
 +
 +
We have chosen LacZ and GFP as the most suitable reporters for our project. The advantage of LacZ compared to GFP is that the reaction of X-Gal with LacZ produces a blue colour which is visible to the naked eye. GFP however needs for its activation light of defined wavelength (488 nm for GFP) and its detection requires complicated Measuring systems. (Fluorescence microscope or FAC-Scan) For the cloning of our constructs, we used Parts from the iGEM registry part and and aplied the iGEM Standard Assembly. Our first clonings were: Sula promoter with lacZ and GFP reporter, as well as RecA promoter with lacZ and GFP reporter.
 +
The plasmid is transformed into a wild type of E. coli.  Now, when DNA is damaged, apart from the normal SOS-Response activation, also the reporter on the synthetic plasmid is activated causing the synthesis of the enzyme. The protein synthesis stars and either GFP or LacZ is produced and can be detected.
 +
After a series of experiments we positive tested our first constructs. Now they showed that there is radiation bit how much radiation.
 +
Our idea was to use recB and recC. These genes are from the same family as RecA, but are activated at different strengths of DNA damage. If we put e.g. three tube next to each other, each containing a construct only with recA, recB or recC. At a particular UV intensity or after a time period of exposure to sunlight recA, B and C are differently activated and show different saturation of blue colour. This graduation in colour can be used to determine the precise amount of radiation. Therefore the UV indicating bacteria are the alternative to the classical Geiger counter.
 +
Now we have this amazing idea and the implementing works well under laboratory conditions, but in which way can we bring it to the consumers. You could carry the bacteria like a plaster on your arm, but this in not practical because if you want to get a tan, there will be allways a white stripe. Therefore we designed a jewellery collection with a tube of our bacteria. Bracelets are small, easy to carry with you and look good.
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Revision as of 17:54, 12 June 2012

iGEM-2012HS - LSL-Heidelberg iGEM-2012HS - LSL-Heidelberg

Introduction

Introduction: The basic idea of our project is to create a simple and cheap tool to measure UV-/ radioactive-mediated damage in living cells. We developed a biological system in E. coli which can react even to different quantities of UV-radiation. The grade of induced damage can be estimated by means of a colorful change of the bacterial suspension. This can be achieved by using the natural SOS response of E. coli. The SOS response is the universe Repair system of prokaryotic cells that response on DNA damage. These mechanisms are very efficient, because they act immediately after the DNA was damaged and repair precisely. This is important, because even small changes in the DNA can have serious consequences. Because of the efficiency the mechanisms can be a high sensitive detector of radiation for us. The the mechanism of the SOS response is further explained here. We have made a construct using a synthetic plasmid backbone with an ORI-region and a selection marker and inserted one of the promoters of a repair protein, followed by the sequence of an enzyme for the colour reaction. Have have chosen to start with the promoter RecA and SulA. These promoters where the most suitable for our project. We have listed all advantages and disadvantages for us of both promoters:

RecA: Recombines Enzym (DNA strand exchange and recombination protein with protease and nuclease activity) Advantages: • Standard gene for SOS-Response • researched and positive tested by other teams • easy to get in the partsregistry: http://partsregistry.org/wiki/index.php/Part:BBa_K154000 • Graduation with recB, recC etc possible Disadvantages: • recA carries out several functions in the E.coli, thus it may be activated even if no radiation is present - might cause disturbance in our data

SulA: SOS cell division inhibitor Advantages: • is only activated for the SOS-response - data would be correct • too short for partsregistry but the oligos can be easily designed and ordered by using the gene code from the partsregistry http://partsregistry.org/wiki/index.php/Part:BBa_K518010 • used by other iGEM teams Disadvantages: not sure if graduations sulB, sulC etc will work for us

We have chosen LacZ and GFP as the most suitable reporters for our project. The advantage of LacZ compared to GFP is that the reaction of X-Gal with LacZ produces a blue colour which is visible to the naked eye. GFP however needs for its activation light of defined wavelength (488 nm for GFP) and its detection requires complicated Measuring systems. (Fluorescence microscope or FAC-Scan) For the cloning of our constructs, we used Parts from the iGEM registry part and and aplied the iGEM Standard Assembly. Our first clonings were: Sula promoter with lacZ and GFP reporter, as well as RecA promoter with lacZ and GFP reporter. The plasmid is transformed into a wild type of E. coli. Now, when DNA is damaged, apart from the normal SOS-Response activation, also the reporter on the synthetic plasmid is activated causing the synthesis of the enzyme. The protein synthesis stars and either GFP or LacZ is produced and can be detected. After a series of experiments we positive tested our first constructs. Now they showed that there is radiation bit how much radiation. Our idea was to use recB and recC. These genes are from the same family as RecA, but are activated at different strengths of DNA damage. If we put e.g. three tube next to each other, each containing a construct only with recA, recB or recC. At a particular UV intensity or after a time period of exposure to sunlight recA, B and C are differently activated and show different saturation of blue colour. This graduation in colour can be used to determine the precise amount of radiation. Therefore the UV indicating bacteria are the alternative to the classical Geiger counter. Now we have this amazing idea and the implementing works well under laboratory conditions, but in which way can we bring it to the consumers. You could carry the bacteria like a plaster on your arm, but this in not practical because if you want to get a tan, there will be allways a white stripe. Therefore we designed a jewellery collection with a tube of our bacteria. Bracelets are small, easy to carry with you and look good.