Team:Heidelberg LSL/Project UVsensors

From 2012hs.igem.org

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<p>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 <a href="http://2012hs.igem.org/Team:Heidelberg_LSL/Notebook">notebook</a>.</p>
<p>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 <a href="http://2012hs.igem.org/Team:Heidelberg_LSL/Notebook">notebook</a>.</p>
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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.
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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/bitstream/handle/1721.1/45138/BBFRFC10.txt?sequence=1"> BBa</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 EcoRI and XbaI to allow for ligation with promoters via standard assembly.
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 EcoRI and XbaI to allow for ligation with promoters via standard assembly.

Latest revision as of 10:27, 18 July 2012

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


Sensor Construction

Overview

Sensory Construction Overview

SOS response promoters from E.coli were either annealed from synthetic oligos or taken from the partsregistry. Promoters were ligated together with digested standard reporter constructs 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 BBa 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 EcoRI and XbaI to allow for ligation with promoters 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 backbone.

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

Sensor constructs

Those constructs were successfully generated, characterized and submitted as parts.

Map based on precA_lacZ_pSB1C3.

Map based on precB_lacZ_pSB1C3.

Map based on psulA_lacZ_pSB1C3.