Team:Heidelberg LSL/Project intro

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

Introduction

Introduction: The basic idea of our project was 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 damage induced can be estimated by means of a colour reaction change in the bacterial suspension. To do so we take advantage of the natural SOS response of E.coli. The SOS response is a universal repair system of prokaryotic cells as a response to DNA damage. The mechanisms involved work very efficiently, starting immediately after the DNA was damaged and resulting in precise repair. This is of utmost importance because even small changes (point mutations) in the DNA may have serious consequences. Due to the efficiency of the mechanisms they serve as highly sensitive detector of radiation for us. For details of the mechanism of the SOS response please see here. We 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.

PROMOTERS We choose to start with the promotors RecA and SulA. These promotors deemed most suitable for our project. Advantages and disadvantages of both promotors for our experiments are listed below:

RecA: Recombinase enzyme (DNA strand exchange and recombination protein with protease and nuclease activity) Advantages: • standard gene for SOS-response • well characterized and successful when used by other teams • easily available through partsregistry: http://partsregistry.org/wiki/index.php/Part:BBa_K154000 • recB, recC are members of the same gene family and may serve as possible candidates to achieve a more gradual UV response Disadvantages: recA is required for a number of functions in E.coli, not only as part of the SOS response. Therefore, it may be activated even if no radiation is present and might give nonspecific results.

SulA: SOS cell division inhibitor Advantages: • is only activated as part of the SOS-response - data probably more specific • too short for Partsregistry but 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 graduation using sulB, sulC etc will work for us

REPORTER GENES

We have chosen LacZ and GFP as the most suitable reporter genes for our project. The advantage of LacZ compared to GFP is the reaction of X-Gal with LacZ which produces a blue colour which is easily visible by eye. GFP however needs light of a defined wavelength (488 nm for GFP) for its activation of and its detection requires measuring systems such as fluorescence microscope or FAC-scan.

CLONING AND ASSEMBLY

For construction of our plasmids we used parts from the iGEM registry part and applied the iGEM standard assembly. Our first clonings were: Sula promoter with lacZ and GFP reporter genes as well as RecA promoter with lacZ and GFP reporter genes.

The plasmid was transformed into BL21 strain of E.coli and performed radiation experiments in replicates of three for varying radiation times. In theory radiation should cause - apart from normal SOS-response activation - the activation of the reporter on the synthetic plasmid and subsequently the protein synthesis of the LacZ or GFP enzymes leading to a colour reaction.

Assuming the system would work under laboratory conditions the ultimate goal of our project is its application as an easy detector of harmful sun radiation in everyday life. We developed ideas how to use bacterial suspensions in a way that is acceptable for normal consumers. One approach was to develop a plaster but was given up because it would leave a white stripe on the skin when sunbathing. As more promising idea is to develop some kind of jewellery containing the bacterial solution such as a bracelet with a little vial.

@@ Line 24: / Line 24: @@
 Introduction:
-The basic idea of our project is to create a simple and cheap tool to measure UV-/ radioactive-mediated damage in living cells.
+The basic idea of our project was 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.
+We developed a biological system in E.coli which can react even to different quantities of UV-radiation. The grade of damage induced can be estimated by means of a colour reaction change in 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 [[link|here]].
+To do so we take advantage of the natural SOS response of E.coli. The SOS response is a universal repair system of prokaryotic cells as a response to DNA damage. The  mechanisms involved work very efficiently, starting immediately after the DNA was damaged and resulting in precise repair. This is of utmost importance because even small changes  (point mutations) in the DNA may have serious consequences. Due to the efficiency of the mechanisms they serve as highly sensitive detector of radiation for us. For details of the mechanism of the SOS response please see 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.
+We 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:
+PROMOTERS
+We choose to start with the promotors RecA and SulA. These promotors deemed most suitable for our project. Advantages and disadvantages of both promotors for our experiments are listed below:
 RecA:
-Recombines Enzym (DNA strand exchange and recombination protein with protease and nuclease activity)
+Recombinase enzyme (DNA strand exchange and recombination protein with protease and nuclease activity)
 Advantages:
-• Standard gene for SOS-Response
+• standard gene for SOS-response
-• researched and positive tested by other teams
+• well characterized and successful when used by other teams
-• easy to get in the partsregistry: http://partsregistry.org/wiki/index.php/Part:BBa_K154000
+• easily available through partsregistry: http://partsregistry.org/wiki/index.php/Part:BBa_K154000
-• Graduation with recB, recC etc possible
+• recB, recC are members of the same gene family and may serve as possible candidates to achieve a more gradual UV response
 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
+recA is required for a number of functions in E.coli, not only as part of the SOS response. Therefore, it may be activated even if no radiation is present and might give nonspecific results.
 SulA:
 SOS cell division inhibitor
 Advantages:
-• is only activated for the SOS-response - data would be correct
+• is only activated as part of the SOS-response - data probably more specific
-• 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
+• too short for Partsregistry but 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
+not sure if graduation using sulB, sulC etc will work for us
+REPORTER GENES
+We have chosen LacZ and GFP as the most suitable reporter genes for our project. The advantage of LacZ compared to GFP is the reaction of X-Gal with LacZ which produces a blue colour which is easily visible by eye. GFP however needs light of a defined wavelength (488 nm for GFP) for its activation of  and its detection requires measuring systems such as fluorescence microscope or FAC-scan.
+CLONING AND ASSEMBLY
+For construction of our plasmids we used parts from the iGEM registry part and applied the iGEM standard assembly. Our first clonings were: Sula promoter with lacZ and GFP reporter genes as well as RecA promoter with lacZ and GFP reporter genes.
+The plasmid was transformed into BL21 strain of E.coli and performed radiation experiments in replicates of three for varying radiation times. In theory radiation should cause  - apart from normal SOS-response activation - the activation of the reporter on the synthetic plasmid and subsequently the protein synthesis of the LacZ or GFP enzymes leading to a colour reaction.
-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.
+Assuming the system would work under laboratory conditions the ultimate goal of our project is its application as an easy detector of harmful sun radiation in everyday life. We developed ideas how to use bacterial suspensions in a way that is acceptable for normal consumers. One approach was to develop a plaster but was given up because it would leave a white stripe on the skin when sunbathing. As more promising idea is to develop some kind of jewellery containing the bacterial solution such as a bracelet with a little vial.
-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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