Part:BBa_K1916116

Ara-Inducible CBCR6 Expression System

A GAF domain of a cyanobacteriochrome (CBCR) designated CBCR6, found through metagenomic mining and predicted to appear blue by the 2016 Davis team's CBCR color predictive program, with an intein/chitin-binding domain fused to the C-terminus for purification. Expression regulated by a pBAD promoter. It was confirmed after co-transformation and expression that CBCR6 requires a phycocyanobilin co-factor, and must therefore be expressed in a system with heme oxygenase (BBa_I15008) and PcyA (BBa_I15009). It was discovered that CBCR6 cells express very well in E. coli and appear dark blue/purple under normal light, thus confirming the initial program prediction.

Characterization

The ara-inducible system was cloned into pSB1C3 and co-transformed into DH5-alpha cells along with a plasmid containing heme oxygenase and PcyA. After growing the cells to OD=0.9-1 in Terrific Broth, the cultures were induced to a final culture concentration of 1mM arabinose to study the color of the cells once harvested (Fig.1).

Fig.1 Cells expressing CBCR6 spun down 4 hrs after induction.

Fig.2 Absorbance spectra of the CBCR6 protein extracted from the cells. Peak absorbance occurs around 640nm.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 131
  Illegal SpeI site found at 692
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 125
  Illegal SpeI site found at 692
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 65
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 131
  Illegal SpeI site found at 692
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 131
  Illegal SpeI site found at 692
  Illegal NgoMIV site found at 1188
  Illegal AgeI site found at 1278
  
• 1000
  COMPATIBLE WITH RFC[1000]