Team:EPF-Lausanne/LOVTAP

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 (AsLOV2). Absorption of a photon by AsLOV2 triggers the formation of a covalent adduct between FMN (the flavin mononucleotide) cofactor and a conserved cysteine residue. This formation leads to the displacement and unfolding of an helix in the LOV domain, which is likely to mediate a signal propagation.

The output module was the bacterial transcription factor trp repressor (TrpR). TrpR can bind its operator DNA as a homodimer.

By ligating AsLOV2 to TrpR, they were able to construc an allosteric switch called LovTAP : LOV- and tryptophan-activated protein. This protein protects DNA when illuminated.

LovTAP can thus be found in two states : dark-state and light-state. The TrpR domain associate with the shared helix (shared from AsLOV2 and TrpR). In the dark state, a few residues of the share helix presumably dissociate from the TrpR domain and dock against the LOV domain. The steric overlap is then relieved, which decreases the TrpR domain's DNA-binding affinity (when the shared helix contacts the LOV domain, the TrpR domain is in an inactive conformation). In the light state, the residues reassociate with the TrpR domains (because the photoexcitation disrupts contacts between the shared helix and the LOV domain), which restores DNA-binding affinity and the system is in the active form. LovTAP can then bind DNA. But this system is not stable, and the LOV domains returns to the dark state, which triggers the dissociation of LovTAP from the DNA).

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