Team:Heidelberg LSL/Project intro

From 2012hs.igem.org

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Revision as of 19:21, 12 June 2012

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

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.

Promoter Genes

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:

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.