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LOVTAP
From 2009.igem.org
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:Direct mutational assay: Insert mutation in specific sites thanks to the known structure of LOVTAP (already modeled on computer) | :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 | :Indirect mutational assay: Random mutations, then selection of the "right" protein according to a set of selected conditions | ||
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<i><font size=3> in vivo </font size></i> | <i><font size=3> in vivo </font size></i> | ||
Revision as of 20:18, 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
- 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 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
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
- </b>
- 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
in vivo
- Express LOVTAP under control of an inducible promoter
- Link a reporter cassette with TrpR binding domain
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