Team:Heidelberg LSL/Project UVsensors

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Strategy

Sensor Construction

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 constructs.

Description

It was intended to generate constructs that are able to sense irradiation and respond to UV light with a colorful output. For details of the cloning procedure, please refer to our notebook.

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 precA as biobrick BBa_J22106. We designed the oligos for precB, precC and psulA 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 precA part was therefore digested with EcoRI and SpeI.
As reporters, we decided for GFP and LacZ from the biobricks BBa_E0240 and BBa_K173004, respectively. We digested those plasmids with XbaI and PStI to allow for ligation with promoters into backbone pSB1K3 via standard assembly.
Finally, constructs were transformed into E. coli strain BL21 and applied to sense UV radiation with visible output. Prior to submission, parts were transferred into pSB1C3.

Details

The following constructs were created by the above explained strategy.

Number	Promoter	Reporter	Backbone
01	precA	gfp	pSB1K3
02	psulA	gfp	pSB1K3
03	precA	lacZ	pSB1K3
04	precB	lacZ	pSB1K3
05	precC	lacZ	pSB1K3
06	psulA	lacZ	pSB1K3
07	precA	lacZ	pSB1C3
08	precB	lacZ	pSB1C3
09	psulA	lacZ	pSB1C3