Part:BBa_K2406079

Lox-Term-Rox Measurement Construct

Introduction

This measurement construct was used to test the cross-reactivity of Lox and Rox (BBa_K1680005, BBa_K2406000). The theory behind the function of this measurement construct is summarised in the adjacent figure. Essentially, when two recombination sites cannot be recognised by a single recombinase, the terminator (represented as parallel lines in the diagram) will not be excised and there will be no RFP reporter outlook. This part is useful because it tests the cross-reactivity of the target sites in question. In order to catalyse two independent, distinct recombination events in one cell with two recombinase systems, it is vital that there is no cross-reactivity. Thus, this measurement construct tests the suitability for using Cre/LoxP and Dre/Rox in one cell.

Schematic outlining principle of all measurement constructs used by Edinburgh_UG 2017

Results

All assays performed using this measurement construct are summarised to the right. For reference, cross-reactivity and fluorescence output is compared to other measurement constructs in the context of Dre BBa_K2406081 and Rox BBa_K2406000. We observed no cross-reactivity within this construct, as fluorescence output was negligible when Dre waspresent and not present.

All measurements performed involving Rox target sites

All measurements performed involving Dre

Discussion

The target sites involved in this construct were previously discovered as being potentially orthogonal to other recombinases [1]. It is therefore important to test their cross-reactivity extensively to fully understand what recombinases can be used within one cell. This had not been done extensively before. Our results demonstrate no cross reactivity between the two target sites, BBa_K1680005, BBa_K2406000. This is because negligible RFP output was seen compared to control. Therefore, the Cre/LoxP and Dre/Rox can be used in an orthogonal manner 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 886
  Illegal AgeI site found at 998
  
• 1000
  COMPATIBLE WITH RFC[1000]