Part:BBa_K3634009

ccaS + ho1 + pcyA (Optimised for E.coli)

The membrane-associated histidine kinase ccaS is part of the two-component system (TCS) involved in the eventual transcriptional output of an adjacent phycobilisome-related gene (cpcG2). The system is native to Synechocystis sp. PCC6803 but has been successfully expressed in E.coli (Hirose et al. 2008) and has been further used in multichromatic control of gene expression (Tabor et al. 2011).

CcaS, alongside the response regulator ccaR, functions as a photoreversible switch between green (535nm) and red (672nm) light by regulation of the output promoter PcpcG2. Within the N-terminal GAF domain of ccaS, the blue phycobilin chromophore phycocyanobilin (PCB) binds to a conserved cysteine residue, imparting reversible photoactivation of signalling activity. Absorption of green light increases the rate of ccaS autophosphorylation, phosphotransfer to ccaR and transcription from PcpcG2. Absorption of red light by ccaS is shown to reverse the process and therefore reduce expression of the output gene. CcaS is also shown to be inactive in the dark. The required PCB chromophore is produced from heme by the following two enzymes.

In the presence of oxygen, heme oxygenase (ho1) catalyses the opening of the heme ring, releasing iron and generating biliverdin IXα. The second enzyme, phycocyanobilin:ferredoxin oxidoreductase (pcyA), then catalyses the four-electron reduction of biliverdin IXα to PCB. PCB then binds to the N-terminal GAF domain of ccaS, allowing for transcriptional output of the gene of interest.

BBa_K3634006, BBa_K3634007 and BBa_K3634008 are combined here as a composite part with the associated regulatory regions used by Schmidl et al. (2014). The system they produced, termed CcaSR v2, uses two plasmids for constitutive expression of ccaS and ccaR, and optimised promoter and RBS combinations to maximise PCB production. The design will be recreated in composite BioBrick part form using E.coli codon optimised parts to further promote efficiency of the system.

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
  Illegal NheI site found at 2449
  Illegal NheI site found at 2472
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2126
  Illegal BglII site found at 2879
  Illegal BamHI site found at 1465
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1497
  
• 1000
  COMPATIBLE WITH RFC[1000]