Difference between revisions of "Part:BBa K2406001"

Latest revision as of 22:05, 28 October 2017

Vox (Target site for Vika)

Introduction

Vika recombinase BBa_K2406082 is a tyrosine recombinase that catalyses recombination between two Vox 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 that Vox can recombine with itself when Vika recombinase BBa_K2406082 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. We tested Vox self-recognition using BBa_K2406053. We observed recombination with self. This is based on high observed RFP fluorescence output. Cross reactivity was observed with target site Vlox BBa_K2406002, which produced less fluorescent output as the self-self construct. This indicates lower efficiency of recombination, but still significant cross-reactivity.

All measurements taken in constructs containing Vox target sites

Discussion

Our results demonstrate that recombination can efficiently occur between two Vox sites. We observed little cross reactivity with target sites Rox and LoxP (<partinfoBBa_K2406000</partinfo>, BBa_K1680005. Surprisingly, we observed cross reactivity with other target sites Vlox <partinof>BBa_K2406002</partinfo> that was not known to be non-orthogonal to Vox [1]. This indicates that this target site can be used in an orthogonal manner to Rox and LoxP but cross-react with Vlox. The orthogonal target sites are therefore the only target sites that can be used in combination with Vox to catalyse distinct, orthogonal recombination events dynamically within a single cell.

References

[1]Karimova, M., Abi-Ghanem, J., Berger, N., Surendranath, V., Pisabarro, M.T., Buchholz, F. 2013 “Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system”. Nucleic Acids Research 41(2):e37.

Sequences

File below confirms sequence of all target sites, generators and measurement constructs used. Media:File:Sequencing Results Edinburgh UG.zip

Sequence and Features