Team:Heidelberg LSL/Project intro


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


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.

Promoter Genes

We chose to start with the promotors RecA and SulA. These promotors seemed most suitable for our project.
RecA is a recombinase enzyme, i.e. a DNA strand exchange and recombination protein with protease and nuclease activity. It is the standard gene for the SOS-response therefore it is well characterized. Other scientist have been successful when using recA for gene regulation. It is easily available through partsregistry: Part:BBa_K154000

There is also the possibility to use different members of the same gene family besides recA,  namely recB and recC. This is important for a further approach by using these genes to achieve a more gradual UV response for the detection of qualitative UV-intensity. Our assumption was that  different rec genes are activated based on a certain characteristic extension of DNA damage dependent on UV radiation.
The disadvantage of recA was that 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. If this could be a problem has to be determine in the experiments.

SulA is a protein that acts as a cell division inhibitor during SOS-response. SulA overcomes the disadvantage of recA, because it is only activated as part of the SOS-response, thus the data would be probably more specific. Although it is too short for Partsregistry but oligos can be easily designed and ordered by using the gene code from the Partsregistry: Part:BBa_K518010
As recA it has also used by other iGEM teams. But for sulA it is if graduation using sulB, sulC etc will work for us to achieve the qualitative detection of UV-intensity.

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 that can easily be seen by eye. GFP however needs excitation-light of a certain wavelength (~488 nm for GFP) in order to emit green light 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 the sulA promoter with lacZ and GFP reporter genes as well as the 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.

Having shown that the system works under laboratory conditions the ultimate goal of our project is its application as an easy detector of harmful UV-radiation emitted by the sun 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 it was rejected because of possible white stripes on the skin when sunbathing. The most promising idea is to develop some kind of jewellery containing the bacterial solution such as a bracelet with a little vial. That is how we invented iGEMS!