Part:BBa_K1916102

Ara-Inducible Cyan7427_1390g3 Expression System

A GAF domain of the cyanobacteriochrome (CBCR) Cyan7427_1390 with an intein/chitin-binding domain fused to the C-terminus for purification. Expression regulated by a pBAD promoter. As with most CBCRs, Cyan7427 requires a phycocyanobilin co-factor, and must therefore be expressed in a system with heme oxygenase (BBa_I15008) and PcyA (BBa_I15009). Cyan7427_1390g3 expresses well in E. coli and cells appear vibrant blue-green under normal light.

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 with increasing levels of arabinose to study the vibrancy of the cells once harvested (Fig.1).

Fig.1 Pellets of cells expressing Cyan7427, spun down 4 hrs after being induced by different concentrations of arabinose.

Fig.2 Absorbance spectra of the Cyan protein extracted from the cells. Peak absorbance occurs around 695nm.

Sequence and Features

Assembly Compatibility:

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

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

  Illegal BamHI site found at 65
  Illegal BamHI site found at 731
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1251
  Illegal AgeI site found at 1341
  
• 1000
  COMPATIBLE WITH RFC[1000]