Part:BBa_K2406003
Slox (Target Site for SCre)
Introduction
Slox Recombinase BBa_K2406084 is a tyrosine recombinase that catalyses recombination between two slox target 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 Slox target site can recombine with itself when Slox Recombinase BBa_K2406084 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.
Results
Our results are summarised on the adjacent figure. We tested Slox self-reactivity using BBa_K2406065. We observed recombination with self. This is based on high observed RFP fluorescence output. Cross reactivity was observed with target site Vlox BBa_K2406002 at a much lower efficiency than self-reactivity.
Discussion
Our results demonstrate that recombination can efficiently occur between two Slox target sites. We observed little cross reactivity with target site Rox BBa_K2406000. Surprisingly, we observed cross reactivity with target site Vlox <partinof>BBa_K2406002</partinfo> that was supposed to be orthogonal with this target site [1]. This indicates that this target site can be used in an orthogonal manner to Rox but cross-react with Vlox at a very low, but potentially significant efficiency. The orthogonal target site Rox is therefore the only target site that can be used in combination with Slox to catalyse distinct, orthogonal recombination events dynamically within a single cell.
References
[1]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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |