Part:BBa_K2406000
Rox (Dre target site)
Introduction
Dre Recombinase BBa_K2406081 is a tyrosine recombinase that catalyses recombination between Rox 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 Rox can recombine with itself when Dre recombinase BBa_K2406081 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 in all domains of life.
Results
Our results are summarised on the adjacent figure. We tested Rox self-recognition using BBa_K2406051. We observed recombination with self. This is based on high observed RFP fluorescence output. No cross reactivity with other recombinase target sites was observed, as demonstrated on the graph.
Discussion
Our results demonstrate that recombination can efficiently occur between two Rox sites. We observed little cross reactivity with target sites Vox, Slox and Vlox (BBa_K2406001, BBa_K2406003, BBa_K2406002). Therefore, all of these target sites and associated recombinases could be used to catalyse distinct, orthogonal recombination events dynamically within a single cell.
References
[1]Anastassiadis, K., Fu, J., Patsch, C., Hu, S., Weidlich, S., Duerschke, K., Buchholz, F., Edenhofer, F., and Stewart A.F. 2009. “Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice.” Disease Models and Mechanisms: Sep-Oct; 2(9-10):508-515.
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 |