Part:BBa_K649202

PlacIQ-lox71-rfp-lox66-gfp

The lox sequences, lox71 and lox66, have 5 bp on the 5 and 3 ends changed, respectively. DNA segment flanked by lox71 and lox66 marks the point which the enzyme Cre will excise. This Part is expected to express GFP when the lox sites are excised and RFP when they are not. The Cre-mediated recombination of this BioBrick had been studied and proved to be working.

Effect of Cre-meditated recombination at lox71/66 cassette.
The leftmost is a negative control which don't have Cre-expressing plasmid.
The center is an arabinose induced sample which has both Cre plasmid and BioBrick BBa_K649201.
The rightmost is a uninduced strain which has both plasmid like as the center.

In in vivo assay, arabinose induced strain which has Cre-expressing plasmid(PBAD/araC-Cre, BBa_I718008) was expressing GFP, while negative control which doesn't have Cre plasmid was expressing Red florescence. It means that DNA recombination did happen by Cre recombinase. The uninduced sample was supposed to unexpress Cre recombinase, but because of the leaking of PBAD/araC promoter, the excision occured in almost every cell in medium.

For more information, see [http://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#8. our work in Tokyo_Tech 2011 wiki].

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 1016
  Illegal BamHI site found at 14
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 686
  Illegal AgeI site found at 798
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1700