Part:BBa_K2406067

Vox-Term-Vlox Measurement Construct

Introduction

This measurement construct was used to test the cross-reactivity of Vox and Vlox (BBa_K2406001, BBa_K2406002). 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 Vika/Vox and VCre/Vlox 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 Vika and VCre recombinases (BBa_K2406082, BBa_K2406083). We observed unexpected cross-reactivity within this construct, as shown by relatively high fluorescence output when Vox and VCre recombinases (BBa_K2406084, BBa_K2406083) were present.

All assays performed involving Vika

All assays performed involving VCre

Discussion

The target sites involved in this construct were not initially tested together and were potentially ortoghonal [1][2]. However, our cross-reactivity tests in E. coli demonstrate they are not orthogonal, unlike the majority of recombinase target sites we tested. Unexpectedly, we observed cross-reactivity between these two target sites BBa_K2406001, BBa_K2406002. Therefore, this indicates that Vika/Vox and VCre/Vlox cannot be used to catalyse distinct and independent recombination events in one cell.

References

[1]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. [2]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 888
  Illegal AgeI site found at 1000
  
• 1000
  COMPATIBLE WITH RFC[1000]