Team:Heidelberg LSL/Project UVsensors

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Strategy

Overview

SOS response promoters from E. coli were either annealed from synthetic oligos or taken from the registry. Promoters were ligated together with digested standard reporter constructs into standard backbone thereby generating our final radiation sensor construct.

Description

The intention was to generate constructs that are able to sense irradiation and respond to UV light with a colorful output.

First, we looked for proper E. coli promoters known to be involved in the SOS response. While RecB, RecC and SulA promoters were generated with custom-made oligonucleotides, we took RecA as biobrick BBa_J22106. We designed the oligos for RecB, RecC and SulA to contain BB-2 standard prefix and suffix and putative promoter sequences (approx. 50 bp upstream of the start codon of each gene) taken from the EcoliWiki. Oligos were annealed and then directly used for ligation since they already contained the required overhangs. The RecA part was therefore digested with EcoRI and SpeI.
As reporters we used GFP and LacZ from the biobricks BBa_E0240 and BBa_K173004, respectively.