Part:BBa_K2116053

CAT-UPRT Fusion with sfGFP

Characterisation of the CAT-UPRT fusion protein

The response of the dual selector plasmid containing a CAT-UPRT fusion protein (CAT: chloramphenicol acetyltransferase, UPRT: uracil phosphoribosyltransferase) towards 5-fluorouracil was tested. The Keio-Collection strain JW2483-1 (Δuprt) was transformed with a plasmid constitutively expressing the fusion protein. The cells were grown overnight in M9 medium, diluted to OD=0.1 and transferred into a flat bottom 96-well plate. They were grown for another three hours and finally different concentrations of 5-fluorouracil and chloramphenicol were added (10 µl into 190 µl of bacterial culture, final concentration are shown.)
Whereas cells expressing the fusion protein are resistant against chloramphenicol, they are especially sensitive towards 5-fluorouracil.
The cells lacking the plasmid as well as do not have a chromosomal copy of uprt are less inhibited by 5-FU but are sensitive towards chloramphenicol.

Negative control: Response of the uprt-deficient strain JW2483-1 towards different concentrations of 5-fluorouracil and chloramphenicol

CAT-UPRT Fusion Protein: Response of the uprt-deficient strain JW2483-1 constitutively expressing the dual-selector fusion protein towards different concentrations of 5-fluorouracil and chloramphenicol

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 1740
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1060
  
• 1000
  COMPATIBLE WITH RFC[1000]