Part:BBa_K2406051

Rox-Term-Rox Measurement construct.

Introduction

This measurement construct was used to test the self-reactivity of Rox 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 self-reactivity of Rox, verifying that our recombinase generator BBa_K2406081 and Rox target site BBa_K2406000 function as a recombinase/target site system. Thus, this measurement construct demonstrates that Dre/Rox functions in E. coli

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 recombinaseBBa_K2406081 and Rox target sites BBa_K2406000. We observed expected reactivity within this construct, as fluorescence output was observed when the Dre recombinase was present, as induced by a pulse of IPTG.

All assays performed involving Dre recombinase

All assays performed involving Rox target sites

Discussion

The target sites involved in this construct were previously identified as working in a site-specific manner [1]. This test verifies their function as biobrick parts used in E. coli. Our results show that the measurement construct works as expected, confirming the activity of Dre recombinaseBBa_K2406081 and its associated target site Rox BBa_K2406000. Therefore, this measurement construct confirmed the activity of these parts and demonstrated that they are suitable for carrying out a site-specific reaction within cells.

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 876
  Illegal AgeI site found at 988
  
• 1000
  COMPATIBLE WITH RFC[1000]