Difference between revisions of "Part:BBa K2406083"

Latest revision as of 20:16, 28 October 2017

VCre Recombinase Inducible Generator

Introduction

VCre is a tyrosine recombinase that catalyses recombination between Vlox [1]. This can lead to integration, excision, or inversion of the DNA sequence in between these target sites. This is an example of a site-specific recombinase (SSR). SSRs have long been recognised to be excellent biological tools, used in conditional gene knock-outs and dynamic events to change gene expression in cells [1]. Therefore, we sought to create a toolkit of these recombinase parts, the fundamental units of which are recombinase generators. Here, Vlox is under the control of our inducible T7 promoter BBa_K2406020. We then tested its activity using our measurement constructs, described in the adjacent figure. Essentially, a terminator was flanked by two recombinase target sites. When the recombinase could recognise both sites, it recombination would occur and the terminator would be excised, producing RFP output. This test was useful for two reasons. For one, it demonstrated that our recombinase proteins could work, as they would excise their associated target sites. Also, it demonstrated which target sites the recombinase would recognise that were not formally associated with it. This is important because researchers have claimed this recombinase is orthogonal to other popular tyrosine recombinases [1], but this has not been extensively tested.

Schematic outlining principle of all measurement constructs used by Edinburgh_UG 2017

Results

Results are summarised in the adjacent figure. Despite not having time to test expected VCre activity in verification construct BBa_K2406059, we verified that VCre recombinase did have intrinsic recombinase activity due to unexpected cross-reactivity. Little cross-reactivity was observed with Vox or Rox (BBa_K2406001, BBa_K2406000 when tested on measurement constructs BBa_K2406057 and BBa_K2406077. However, some cross-reactivity was observed when tested on measurement constructs BBa_K2406063 and BBa_K2406077, indicating cross reactivity with Slox BBa_K2406003 and LoxP BBa_K1680005.

All assays performed with VCre recombinase

Discussion

We have demonstrated our VCre performs recombinase due to cross-reactivity with Lox and Slox target sites through tests in constructs BBa_K2406063 and BBa_K2406077. RFP output when recombination occurs is much higher than output when there is no recombination when target sites are orthogonal. RFP output in observed in the un-induced control can be explained by leakiness inherent to the T7-LacO promoter we used, BBa_K2406020. This indicates VCre recombinase cannot be used within one cell with Cre BBa_K2406080 or SCre BBa_K2406084 to catalyse distinct recombination events. However, there was no observed cross-reactivity with other target sites. This confirms that the VCre, Vika BBa_K2406082, and Dre BBa_K2406081 can be used in an orthogonal manner in one cell. This opens up exciting possibilities for multiple, dynamic gene editing events within one cell.

References

Suzuki E. and Nakayama, M. 2011. “VCre/VloxP and SCre/SloxP: new site-specific recombination systems for genome engineering.” Nucleic Acids Research 39(8):e49.

Sequences

File below confirms sequences of generator and all test constructs used. Media:File:Sequencing Results Edinburgh UG.zip

Sequence and Features