Team:EPF-Lausanne/LOVTAP
From 2009.igem.org
(New page: {{EPF-Lausanne09}} <div CLASS="epfltrick">__TOC__ </div> <div CLASS="epfl09"> ==To get== <font size=3> '''URGENT: get starting material''' </font size> <br><i><font size=3> in vitro </fo...) |
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- Sequence of the 12 mutant fusion proteins or LOV domain AND TrpR (separately) => [http://www.ncbi.nlm.nih.gov/ Pubmed] | - Sequence of the 12 mutant fusion proteins or LOV domain AND TrpR (separately) => [http://www.ncbi.nlm.nih.gov/ Pubmed] | ||
- | <br> - Purification kit&Digestion assay protocol => find the purification protocols etc. in the supplementary material, not by asking the authors | + | <br> - Purification kit&Digestion assay protocol => find the purification protocols etc. in the supplementary material, not by asking the authors</br> |
- | <br> - LED/light sources or photometer | + | <br> - LED/light sources or photometer</br> |
- | <br> - Calmodulin kit or stuffs for protection assay | + | <br> - Calmodulin kit or stuffs for protection assay</br> |
<i><font size=3> in vivo </font size></i> | <i><font size=3> in vivo </font size></i> | ||
- | <br> - Inducible promoter: IPTG or Lac repressor | + | <br> - Inducible promoter: IPTG or Lac repressor</br> |
- | <br> - Reporter cassette: mCherry or RFP | + | <br> - Reporter cassette: mCherry or RFP</br> |
==To do: theory== | ==To do: theory== | ||
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;- Biobricks | ;- Biobricks | ||
Look for a (or many) paper(s) that characterizes E.Coli Trp repressor, and find the Trp operon sequence <font size=3><span style="color:orange"> Mél </span></font size> | Look for a (or many) paper(s) that characterizes E.Coli Trp repressor, and find the Trp operon sequence <font size=3><span style="color:orange"> Mél </span></font size> | ||
- | <br> Summary of what characterizes E.Coli Trp repressor : [[Media:The_tryptophan_biosynthetic_pathway.pdf]] | + | <br> Summary of what characterizes E.Coli Trp repressor : [[Media:The_tryptophan_biosynthetic_pathway.pdf]]</br> |
- | <br> One good article : [[RNA-based_regulation_of_genes_of_tryptophan_synthesis_an_degradation.pdf]] | + | <br> One good article : [[RNA-based_regulation_of_genes_of_tryptophan_synthesis_an_degradation.pdf]]</br> |
- | <br> Protein sequence from NCBI : [[Media:Sequence_du_Trp_repressor.txt]] | + | <br> Protein sequence from NCBI : [[Media:Sequence_du_Trp_repressor.txt]]</br> |
- | <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. | + | <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.</br> |
==To do: wet lab== | ==To do: wet lab== | ||
- | <br><i><font size=3> in vitro </font size></i> | + | <br><i><font size=3> in vitro </font size></i></br> |
;- Redo the experiment they did in the LOVTAP article: | ;- Redo the experiment they did in the LOVTAP article: | ||
Revision as of 12:08, 6 July 2009
Contents |
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</br>
- LED/light sources or photometer</br>
- Calmodulin kit or stuffs for protection assay</br>
in vivo
- Inducible promoter: IPTG or Lac repressor</br>
- Reporter cassette: mCherry or RFP</br>
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</br>
One good article : RNA-based_regulation_of_genes_of_tryptophan_synthesis_an_degradation.pdf</br>
Protein 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.</br>
To do: wet lab
in vitro </br>
- - 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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