Team:Heidelberg LSL/Project Introduction

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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 detect a broad range of UV-radiation doses. The grade of damage induced can be estimated by means of a color reaction in the bacterial suspension.
To do so we take advantage of the natural SOS response of E.coli. The SOS mechanism is a universal repair system in 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 click 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 color 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. The promoter of this gene is easily available through partsregistry: Part:BBa_K154000

There is also the possibility to use different members of the same gene family besides precA, namely precB and precC. This is important for a further approach by using these genes to achieve a more gradual UV response for the detection of UV-intensity. We assumed that increasing DNA damage leads to the successive induction of the different rec promoters.
The disadvantage of precA was that precA 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 precA it has also used by other iGEM teams. But for psulA it is if graduation using psulB, psulC 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 color 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 flow cytometry.

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 color 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 jewelry containing the bacterial solution such as a bracelet with a little vial. That is how we invented iGEMS!

Scientific Background - The SOS response

In order to realize our project, we took advantage of the DNA-repair machinery of the bacterium E.coli, the so-called SOS response system. 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 occuring during strong DNA damages. Activated recA leads to the cleavage of the lexA-repressor protein that normally blocks the RNA-polymerase for transcription of repair proteins. The activated lexA can no longer bind to the DNA strain and allows the production of a cascade of about 20 different repair proteins, amongst others sulA, uvrB and dinF. Another action of the activated 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.

RecA, lexA, sulA and roughly 15 other proteins make up the SOS response. Amongst 20 different promoters we have chosen precA and psulA for our experiments. We used the following criteria for the selection of the optimal promoters:

• They should be involved in only a few processes in the cell to avoid hazardous disorders.
• The promoters should already be well known, tested and available in the parts registry.

Only precA and psulA meet these criteria. Although precA has many functions in the cell it is a standard gene for the SOS response and well characterised. Another desired feature for our project is the ability to detect the amount of UV radiation by changes in the intensity of colour development. Therefore we are trying to use other rec-proteins besides recA such as recB, recC etc. Our hypothesis is that these rec-proteins may be activated at different UV mediated damages.