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Team:IvyTech-South Bend/Project
From 2012.igem.org
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Overall project
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Project Details
Part 2
We compared the sensitivity of J33201 to the Gronigen Team’s K190015 pArs site using the Endy Lab’s expression vector J61002. We loaded J33201 into a kanamycin resistant backbone pSB1K3 and exchanged it with K190015 by perfoming an upstream cut of both to leave the RFP in the J61002 backbone. (Ratio of J33201 to K190015 plasmid DNA was 3:1) If the increased expression of ArsR had the effect of increasing the threshold of the arsenic response any resulting colonies of J33201 in J61002 we predicted would appear white.
Not wanting apparent success to get in our way we hypothesized that the addition of a second ArsR gene under pArs control should further raise the threshold of the device. We therefore added a terminiator to J33201 and coupled it upstream from J33201 and as a control K190015. A down stream cut of K190015 from J661002 expression plasmid yields pArs+RBS+RFP+Term.
The Experiment
June 18, 2012 Week 1. Staging and presentation of project storming to Ivy Tech advisory board. Parts research and descriptions.
June 25, 2012 Week 2. Continuation of parts research including promoters & killswitch screening and selction. Constitutive PTeT promoter, RBS, double terminator.
July 2, 2012 Week 3. Checked cultures and paired plates. Continuation of parts research and construction
