Part:BBa_K4062025

lactose regulated bla, CAP mutation

This device regulates the expression of b-lactamase using environmental lactose concentration and the regulatory components of the lac operon. The lacI biobrick has a mutation in the CAP binding site that deletes the first five bases. This mutation has been shown to significantly reduce the binding of the CAP protein to the CAP site [1]. The mutation was verified by sequencing.

Additionally, the mutation was observed by a lack of color change in an environment with high lactose concentration and low glucose concentration (pictured). Ideally, the high lactose/low glucose culture would elicit a greater color change, since the lac repressor should not be bound to lacO.

Using more concentrated nitrocefin (chromogenic substrate of b-lactamase) and adding a simultaneous glucose/lactose exposure showed results closer to what was expected (pictured). However, these are subjective and need quantification.

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 2914
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1982