Team:Paris Bettencourt/Experiments/pHyperSpank

From 2011.igem.org

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|E. coli containing Hyperspank +RBS+RFP with and whithout IPTG at 37°C
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|E. coli containing Hyperspank +RBS+RFP whithout IPTG at 37°C
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|[[File:minustrans1.jpg|450px|thumb|center|E.coli  at 37°C  (trans image)]]
|[[File:minustrans1.jpg|450px|thumb|center|E.coli  at 37°C  (trans image)]]
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|[[File:minusfluo1.jpg|450px|thumb|center|E.coli  at 37°C (rfp image)]]
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|[[File:minusfluo1.jpg|450px|thumb|center|E.coli  at 37°C negative control (rfp image)]]
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|[[File:minustrans2.jpg|450px|thumb|center|E.coli  negative control at 37°C  (trans image)]]
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|[[File:minustrans2.jpg|450px|thumb|center|E.coli  at 37°C  (trans image)]]
|[[File:minusfluo2.jpg|450px|thumb|center|E.coli  negative control at 37°C  (rfp image)]]
|[[File:minusfluo2.jpg|450px|thumb|center|E.coli  negative control at 37°C  (rfp image)]]
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{| border="1" class="wikitable" style="text-align: center;"
{| border="1" class="wikitable" style="text-align: center;"
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|E. coli containing Hyperspank +RBS+RFP with and whithout IPTG at 37°C
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|E. coli containing Hyperspank +RBS+RFP with IPTG at 37°C
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|[[File:plustran1.jpg|450px|thumb|center|E.coli  at 37°C  (trans image)]]
|[[File:plustran1.jpg|450px|thumb|center|E.coli  at 37°C  (trans image)]]
|[[File:plusfulo1.jpg|450px|thumb|center|E.coli  at 37°C  (rfp image)]]
|[[File:plusfulo1.jpg|450px|thumb|center|E.coli  at 37°C  (rfp image)]]
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|[[File:plustrans2.jpg|450px|thumb|center|E.coli negative control at 37°C  (trans image)]]
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|[[File:plustrans2.jpg|450px|thumb|center|E.coli   at 37°C  (trans image)]]
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|[[File:plusfluo2.jpg|450px|thumb|center|E.coli  negative control at 37°C  (rfp image)]]
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|[[File:plusfluo2.jpg|450px|thumb|center|E.coli  at 37°C  (rfp image)]]
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Revision as of 04:15, 22 September 2011

Team IGEM Paris 2011

pHyperSpank characterisation

The tRNA Amber diffusion system principle

The Hyperspank promoter was designed to be compatible for B. subtilis. It is shorter thant the classic one, but is also recognised by E. coli polymerases.
We cloned it first behind the amber tRNA that would induce a GFP amber on an other plasmid. Then to characterise it we cloned it behind an RFP, into TURBO cells that are LacIq to allow the promoter to be the less leacky possible.
The pictures below are the results of an IPTG induction.

Results

The new biobrick pHS (K606040) was cloned in front of an RFP terminator. The fluorescence was seen under the microscope with strains induced and not induced but IPTG

E. coli containing Hyperspank +RBS+RFP whithout IPTG at 37°C
E.coli at 37°C (trans image)
E.coli at 37°C negative control (rfp image)
E.coli at 37°C (trans image)
E.coli negative control at 37°C (rfp image)
E. coli containing Hyperspank +RBS+RFP with IPTG at 37°C
E.coli at 37°C (trans image)
E.coli at 37°C (rfp image)
E.coli at 37°C (trans image)
E.coli at 37°C (rfp image)

The absolute value of the net fluorescence over OD for induced and non indeuced cells was measured in the TECAN i-control, and plotted below.

Spectrophotometry.jpg




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