Team:Heidelberg LSL/Project UVsensors

From 2012hs.igem.org

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<p>First, we looked for proper <i>E. coli</i> 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 <a href="http://partsregistry.org/Part:BBa_J22106">BBa_J22106</a>. We designed the oligos for precB, precC and psulA to contain <a href="http://dspace.mit.edu/handle/1721.1/45139"> BB-2</a> standard prefix and suffix and putative promoter sequences (approx. 50 bp upstream of the start codon of each gene) taken from the <a href="http://ecoliwiki.net/Main_Page">EcoliWiki</a>. 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.
<p>First, we looked for proper <i>E. coli</i> 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 <a href="http://partsregistry.org/Part:BBa_J22106">BBa_J22106</a>. We designed the oligos for precB, precC and psulA to contain <a href="http://dspace.mit.edu/handle/1721.1/45139"> BB-2</a> standard prefix and suffix and putative promoter sequences (approx. 50 bp upstream of the start codon of each gene) taken from the <a href="http://ecoliwiki.net/Main_Page">EcoliWiki</a>. 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.
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As reporters, we decided for GFP and LacZ from the biobricks <a href="http://partsregistry.org/Part:BBa_E0240">BBa_E0240</a> and <a href="http://partsregistry.org/Part:BBa_K173004">BBa_K173004</a>, respectively. We digested those plasmids with XbaI and PStI to allow for ligation with promoters into backbone <a href="http://partsregistry.org/Part:pSB1K3">pSB1K3</a> via standard assembly.</p>
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As reporters, we decided for GFP and LacZ from the biobricks <a href="http://partsregistry.org/Part:BBa_E0240">BBa_E0240</a> and <a href="http://partsregistry.org/Part:BBa_K173004">BBa_K173004</a>, respectively. We digested those plasmids with XbaI and PStI to allow for ligation with promoters into backbone <a href="http://partsregistry.org/Part:pSB1K3">pSB1K3</a> via standard assembly.
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Finally, constructs were transformed into <i>E. coli</i> strain BL21 and applied to sense UV radiation with visible output.
Finally, constructs were transformed into <i>E. coli</i> strain BL21 and applied to sense UV radiation with visible output.
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Prior to submission, parts were transferred into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>.
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Prior to submission, parts were transferred into <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a>.</p>
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<h4>Details</h4>
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Revision as of 21:35, 16 June 2012

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

Strategy

Overview

Sensory Construction 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 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