Part:BBa_K3809009

mRFP + oxyR homology regions + FLP recognition sequence

This sequence codes for the reporter protein mRFP. It was designed as part of a novel selection method in which the oxidative stress regulator gene (oxyR) is removed. mRFP will act as a reporter and will indicate if the knockout was succesful or not. This gene will work alongside aldO, PuDHT and katE to develop a new selection mechanism which involves the knockout of genes gldA, glpK and oxyR to make E. coli suceptible to death by exposure to low concentrations of glycerol. We will substitute the genes oxyR, gldA and glpK with mRFP, PuDHT and aldO, and transform the bacteria with a plasmid containing selection cassette katE. AldO will convert glycerol to peroxide and D-glyceric acid, katE will convert peroxide to water and oxygen and PuDHT will convert D-glyceric acid into pyruvate. The flippase recognition target region was added to remove the reporter after the knockout of oxyR. However, if the enzyme flippase recombinase isn't present, it will not remove the reporter. It is regulated by BBa_J23100 promoter and has a BBa_B0030 RBS and an rrnB T1 terminator BBa_B0010. It also has a spacer between the promoter and the RBS (BBa_B0040). The reporter was obtained from part BBa_J23100.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]