Part:BBa_K2406053

Vox-Term-Vox Measurement Construct

Introduction

This measurement construct was used to test the self-reactivity of Vox BBa_K2406001. 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 Vox, verifying that our Vika recombinase generator BBa_K2406081 and Vox target site BBa_K2406001 function as a recombinase/target site system. Thus, this measurement construct demonstrates that Vika/vox 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 Vika recombinaseBBa_K2406082 and Vox target sites BBa_K2406001. We observed expected reactivity within this construct, as fluorescence output was observed when the Vikarecombinase was present, as induced by a pulse of IPTG.

All assays performed involving Vika recombinase

All assays performed involving Vox 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 Vika recombinaseBBa_K2406082 and its associated target site Vox BBa_K2406001. 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]Karimova, M., Abi-Ghanem, J., Berger, N., Surendranath, V., Pisabarro, M.T., Buchholz, F. 2013 “Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system”. Nucleic Acids Research 41(2):e37.

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 880
  Illegal AgeI site found at 992
  
• 1000
  COMPATIBLE WITH RFC[1000]