LOVTAP

From 2009.igem.org

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Revision as of 20:06, 3 June 2009

To get

URGENT: get starting material

in vitro

- Sequence of the 12 mutant fusion proteins or LOV domain AND TrpR (separately) => [http://www.ncbi.nlm.nih.gov/ Pubmed]
- Purification kit
- Digestion assay protocol
- LED/light sources or photometer
- Calmodulin kit or stuffs for protection assay

in vivo

To do

- Write an e-mail to Strickland et al to ask Basile first shot; I think we could then look at it all together

letter :Media:letter_T.R.Sosnick.pdf

- Redo the experiment they did in the LOVTAP article: Express and purify mutants; is flavin indispensable??; Trp has to be added

- Find the exact genetic circuit for Trp repressor Nath

An interesting course on TrpR and Trp operon: [http://www2.hawaii.edu/~scallaha/SMCsite/475%20Lectures/475Lecture34.pdf TrpR]

- Biobricks

Look for a (or many) paper(s) that characterizes E.Coli Trp repressor, and find the Trp operon sequence Mél
Summary of what characterizes E.Coli Trp repressor : Media:The_tryptophan_biosynthetic_pathway.pdf
One good article : RNA-based_regulation_of_genes_of_tryptophan_synthesis_an_degradation.pdf‎
Sequence from NCBI : Media:Sequence_du_Trp_repressor.txt‎‎
Design a biobrick that coexpresses LOVTAP and mCherry (after Trp operon) when LOVTAP bind the Trp operon. Design a switch on/off read out.

- Do a mutational assay to change specificity of LOVTAP:

Direct mutational assay: Insert mutation in specific sites thanks to the known structure of LOVTAP (already modeled on computer)

Indirect mutational assay: Random mutations, then selection of the "right" protein according to a set of selected conditions

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 ==To get==
 <b><font size=3> URGENT: get starting material </font size></b>
 <br><i><font size=3> in vitro </font size></i>