Difference between revisions of "Part:BBa K2406081"

Latest revision as of 20:15, 28 October 2017

Dre Recombinase Inducible Generator

Introduction

Dre is a tyrosine recombinase that catalyses recombination between Rox [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, Dre 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. Dre was shown to excise the terminator when it recognised the two Rox sites it was expected to excise on measurement construct BBa_K2406051. Little cross-reactivity was observed with Vox, Slox, or Vlox (BBa_K2406001, BBa_K2406003, BBa_K2406002) when tested on measurement constructs BBa_K2406073, BBa_K2406071, and BBa_K2406077

Results of all measurements involving inducibly expressed Dre recombinase. +IPTG indicates induction

Discussion

We have demonstrated our Dre recombinase part performs the basic recombination event that is expected of it, i.e. it recognises its associated target sites and catalyses recombination between them. RFP output when recombination occurs is much higher than output when there is no recombination or cross-reactivity. RFP output in observed in the un-induced control can be explained by leakiness inherent to the T7-LacO promoter we used, BBa_K2406020. We observed no cross-reactivity with Vox, Slox, or Vlox target sites, tested respectively in BBa_K2406073, BBa_K2406071, BBa_K2406077, indicating that these recombinases can be used within one cell to catalyse distinct recombination events.

References

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

Sequence and Features