Part:BBa_K2406084

SCre Recombinase Inducible generator

Introduction

SCre is a tyrosine recombinase that catalyses recombination between Slox [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, SCre 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. SCre was shown to excise the terminator when it recognised the two Slox target sites it was expected to excise on measurement construct BBa_K2406065. Little cross-reactivity was observed with Vlox BBa_K2406002 or Rox BBa_K2406000 when tested on measurement constructs BBa_K2406063 and BBa_K2406071.

All measurements taken by Edinburgh_UG assessing activity of SCre Recombinase

Discussion

We have demonstrated our SCre recombinase 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. There was no observed cross-reactivity with other target sites. This confirms that the Dre recombinase BBa_K2406081 and Vika recombinase BBa_K2406082 can be used in an orthogonal manner in one cell. This opens up exciting possibilities for multiple, dynamic gene editing events within one 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 sequences of generator and all test constructs used. Media:File:Sequencing Results Edinburgh UG.zip

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1207
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 399
  
• 1000
  COMPATIBLE WITH RFC[1000]