Team:Edinburgh OG/Improve
Improve
INTRODUCTION
A core principle of the Biobrick foundation is to bring standardisation in a field that is constantly changing and evolving. Standardising biological parts and their assembly allows for projects to focus less on assembly of genetic constructs and more on design and testing.
LovTAP is a DNA binding fusion protein between a Light-oxygen-voltage-sensing (LOV2) domain from Avena sativa and tryptophan repressor (TrpR) common in E. coli developed and improved in 2008[1],[2]. The LovTAP fusion protein was initially submitted to the registry by the École Polytechnique Fédérale de Lausanne 2009 iGEM team[3]. The LovTAP fusion protein acts as a photoswitch allowing selective DNA binding through the TrpR domain regulated by the asLOV2 domain. The asLOV2 domain sensitive to photoexitation at 470 nm exists in two conformational; a dark state and light state. Photoexcitation at 470 nm of the LOV domain results in the conformational change of the trpR domain allowing DNA binding, DNA disassociation occurs when the LOV domain returns to the inactive dark state causing the conformation to revert.
IMPROVEMENTS
The original Biobrick BBa_K191006 is only compatible with a single assembly method (RFC 21), limiting availability on the registry and hence hindering further future work. Redesigning of the original brick and optimising the coding sequence for the iGEM registry would result in extended compatibility, facilitating future work and characterisation. Our aim is to create a variation of the original Biobrick completely compatible with all iGEM assembly standards without resulting in loss of function.
The optimised biobrick BBa_K2330002 was initially tested by ligation into plasmid backbone pSB1C3 assembled through the RFC10 standard. Successful ligation was determined through colony PCR. Previous work on the origional LovTAP part indicated that LovTAP under a lacl operator was highly expressed even without IPTG induction. To test experimental activity, the optimised Biobrick was assembled into a low copy number plasmid pSB3K5
[1] Strickland, D. et al. ‘Rationally Improving LOV Domain–based Photoswitches’. Nat. Methods 7, 623–626 (2010).
[2] Strickland, D., Moffat, K. & Sosnick, T. R. ‘Light-Activated DNA Binding in a Designed Allosteric Protein.’ Proc. Natl. Acad. Sci. U. S. A. 105, 10709–14 (2008).
[3] ‘Team:EPF-Lausanne/LOVTAP - 2009.igem.org’. Available at: https://2009.igem.org/Team:EPF-Lausanne/LOVTAP.
