LOVTAP

From 2009.igem.org

(Difference between revisions)

Revision as of 20:10, 3 June 2009

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
- 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

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

in vivo

- Find the exact genetic circuit for Trp repressor Nath

An interesting course on TrpR and Trp operon: 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.

To do: wet lab

- 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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@@ Line 15: / Line 15: @@
 <br> - Reporter cassette mCherry or RFP
-==To do==
+==To do: theory==
 ;- Write an e-mail to Strickland et al to ask<font size=3><span style="color:Blue"> Basile</span></font size> <span style="color:grey">first shot; I think we could then look at it all together</span>
 <span style="color:grey">letter :[[Media:letter_T.R.Sosnick.pdf]]
 <br><i><font size=3> in vitro </font size></i>
@@ Line 24: / Line 25: @@
 : <span style="color:red">is flavin indispensable??
 : Trp has to be added
 <i><font size=3> in vivo </font size></i>
@@ Line 35: / Line 37: @@
 <br> Sequence from NCBI : [[Media:Sequence_du_Trp_repressor.txt‎‎]]
 <br> 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==
 <br><b>- Do a mutational assay to change specificity of LOVTAP:</b>