LOVTAP

From 2009.igem.org

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{{EPF-Lausanne09}}
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==To get==
==To get==
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<b><font size=3> URGENT
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<font size=3> '''URGENT: get starting material''' </font size>
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<br><i><font size=3> in vitro </font size></i>
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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]
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<br> - Purification kit
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<br> - Purification kit&Digestion assay protocol => find the purification protocols etc. in the supplementary material, not by asking the authors
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<br> - Digest assay protocol
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<br> - LED/light sources or photometer
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<br> - Calmodulin kit or stuffs for protection assay
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==To do==
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<i><font size=3> in vivo </font size></i>
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<br> - Inducible promoter: IPTG or Lac repressor
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<br> - Reporter cassette: mCherry or RFP
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==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>
;- 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]]
<span style="color:grey">letter :[[Media:letter_T.R.Sosnick.pdf]]
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;- Redo the experiment they did in the LOVTAP article:
 
-
: Express and purify mutants
 
-
: <span style="color:red">is flavin indispensable??
 
-
: Trp has to be added
 
 +
<i><font size=3> in vitro </font size></i>
 +
 +
<i><font size=3> in vivo </font size></i>
;- Find the exact genetic circuit for Trp repressor <font size=3><span style="color:purple"> Nath </span></font size>
;- Find the exact genetic circuit for Trp repressor <font size=3><span style="color:purple"> Nath </span></font size>
An interesting course on TrpR and Trp operon: [http://www2.hawaii.edu/~scallaha/SMCsite/475%20Lectures/475Lecture34.pdf TrpR]
An interesting course on TrpR and Trp operon: [http://www2.hawaii.edu/~scallaha/SMCsite/475%20Lectures/475Lecture34.pdf TrpR]
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<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> 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]]
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<br> Sequence from NCBI : [[Media:Sequence_du_Trp_repressor.txt‎‎]]
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<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> Design a biobrick that coexpresses  LOVTAP and mCherry (after Trp operon) when LOVTAP bind the Trp operon. Design a switch on/off read out.
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<br><b>- Do a mutational assay to change specificity of LOVTAP:</b>
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==To do: wet lab==
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:Direct mutational assay: Insert mutation in specific sites thanks to the known structure of LOVTAP (already modeled on computer)
+
 
 +
 
 +
<br><i><font size=3> in vitro </font size></i>
 +
;- 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
 +
: <span style="color:red">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
:Indirect mutational assay: Random mutations, then selection of the "right" protein according to a set of selected conditions
 +
: <i>''' In vivo:'''</i> 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
 +
 +
 +
<i><font size=3> in vivo </font size></i>
 +
: Express LOVTAP under control of an inducible promoter
 +
: Link a reporter cassette with TrpR binding domain
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----
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<div CLASS="epfl09bouchon"></div>
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!align="center"|[[Team:EPF-Lausanne|Home]]
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!align="center"|[[Team:EPF-Lausanne/Team|The Team]]
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!align="center"|[[Team:EPF-Lausanne/Project|The Project]]
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!align="center"|[[Team:EPF-Lausanne/Parts|Parts Submitted to the Registry]]
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!align="center"|[[Team:EPF-Lausanne/Modeling|Modeling]]
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!align="center"|[[Team:EPF-Lausanne/Notebook|Notebook]]
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!align="center"|[[Team:EPF-Lausanne/Lectures|Lectures]]
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!align="center"|[[Team:EPF-Lausanne/Team Management|Team Management]]
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Latest revision as of 12:41, 5 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
- 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


</div>

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