Difference between revisions of "Part:BBa K2406002"

Latest revision as of 22:31, 28 October 2017

Vlox (VCre Target Site)

Introduction

VCre recombinase BBa_K2406083 is a tyrosine recombinase that catalyses recombination between two Vlox sites [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, a fundamental unit of which is the associated target sites for each recombinase. Here, we demonstrate unexpected cross-reactivity of this target site to Vox BBa_K2406001 when either associated recombinase is present. We then tested its cross-reactivity potential 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 target sites worked as expected, as they would excise their associated target sites. Also, it demonstrated which target sites unexpectedly could cross-react. This is important because researchers have claimed this target site is orthogonal to other popular target sites [1], but this has not been extensively tested.

Schematic outlining principle of all measurement constructs used by Edinburgh_UG 2017

Results

Our results are summarised on the adjacent figure. Due to time constraints, we could not test self recombination with the construct BBa_K2406059. However, we still saw the that this site functions as a recombinase target site due to unexpected cross-reactivity. Cross reactivity was observed with target site Vox BBa_K2406001 and Slox BBa_K2406003to a much lesser extent in measurement constructs BBa_K2406067 and BBa_K2406063, respectively. The cross reactivity is demonstrated through high RFP output.

All assays carried out by Edinburgh_UG involving Vlox target sites

Discussion

Our results demonstrate that Vlox functions as a target site for VCre recombinase, due to unexpected cross-reactivity with Vox target sites BBa_K2406001. We observed little cross reactivity with target site Rox BBa_K2406000. Surprisingly, we observed cross reactivity with other target site Vox BBa_K2406001 and Slox BBa_K2406003. Lower output to of RFP in the Slox measurement construct could indicate a much lower incidence of cross-reactive recombination due to inherent inefficiencies of the recombination machinery recognising Slox and Vlox. This indicates that this target site can be used in an orthogonal manner to Rox target site but cross-react with Vox and to a lesser extent Slox. The orthogonal target site Ros is therefore the only target site that can be used in combination with Vlox to catalyse distinct, orthogonal recombination events dynamically within a single 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 sequence of all target sites, generators and measurement constructs used. Media:File:Sequencing Results Edinburgh UG.zip

Sequence and Features