LOVTAP

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 => 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

- 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

in vitro

in vivo

- 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
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.

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

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