LOVTAP

To get
URGENT: get starting material

 in vitro 

- Sequence of the 12 mutant fusion proteins or LOV domain AND TrpR (separately) => Pubmed - Purification kit&Digestion assay protocol => find the purification protocols etc. in the supplementary material, not by asking the authors - LED/light sources or photometer - Calmodulin kit or stuffs for protection assay

 in vivo  - Inducible promoter: IPTG or Lac repressor - Reporter cassette: mCherry or RFP

To do: theory
letter :[[Media:letter_T.R.Sosnick.pdf]]
 * - Write an e-mail to Strickland et al to ask Basile first shot; I think we could then look at it all together

 in vitro 

 in vivo  An interesting course on TrpR and Trp operon: TrpR
 * - Find the exact genetic circuit for Trp repressor Nath

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 Protein 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.
 * - Biobricks

To do: wet lab
 in vitro 
 * - Redo the experiment they did in the LOVTAP article:

!!! Major problem, the conformational change of LOVTAP is weak and the protection assay results show a small difference of LOVTAP binding on DNA between drak state and light state !!! > try to improve this
 * Express and purify mutants
 * is flavin indispensable??
 * Trp has to be added


 * - Do a mutational assay to change or enhance specificity of LOVTAP:
 * Directed 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
 *  In vivo: Evolved mutational assay: LovTap inhibit a killer gene, so the more the lovtap affinity for DNA is high the more likely cells will survive (simulation of evolutionnary selection)
 * -> Other in vitro techniques: SELEX

 in vivo 
 * Express LOVTAP under control of an inducible promoter
 * Link a reporter cassette with TrpR binding domain