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Team:EPF-Lausanne/LOVTAP
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==LOVTAP system== | ==LOVTAP system== | ||
| + | In the article of Strickland and al., an allosteric switch was created by joining two domains. The resulting protein has a domain-domain overlap with a shared helix. This shared helix acts as a rigid lever arm. The disruption of the helical contacts causes a shift in the conformation. Thus, photoexcitation would change the conformation of the protein, and that will change the stability of the helix-domain contacts. This will change the affinity of the shared helix for the two domains, and a signal could be then sent. | ||
| + | In the LOVTAP system, the light-sensitive input module is the LOV2 domain of Avena sativa phototropin 1. | ||
| + | ... | ||
==To get== | ==To get== | ||
Revision as of 11:50, 22 July 2009
LOVTAP system
In the article of Strickland and al., an allosteric switch was created by joining two domains. The resulting protein has a domain-domain overlap with a shared helix. This shared helix acts as a rigid lever arm. The disruption of the helical contacts causes a shift in the conformation. Thus, photoexcitation would change the conformation of the protein, and that will change the stability of the helix-domain contacts. This will change the affinity of the shared helix for the two domains, and a signal could be then sent.
In the LOVTAP system, the light-sensitive input module is the LOV2 domain of Avena sativa phototropin 1.
...
To get
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
- Reporter cassette: RFP
To do: theory
in vivo
- - Find the exact genetic circuit for Trp repressor
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 :
Summary of what characterizes E.Coli Trp repressor : Media:The_tryptophan_biosynthetic_pathway.pdf
One good article : Media: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 RFP (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 (2)
!!! 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
