Part:BBa_K3634019

CcaR-Mediated CcdA Expression System

The response regulator ccaR is part of the two-component system (TCS) involved in the eventual transcriptional output of an adjacent phycobilisome-related gene (cpcG2) in response to green light of wavelength 535nm. 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).

CcaR, alongside the membrane-associated histidine kinase ccaS, 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 and phosphotransfer to ccaR. Once phosphotransfer has occurred, ccaR binds to an operator site within the sequence of the output promoter PcpcG2. Transcription of the output gene is then activated.

The natural output gene cpcG2 has in the past been replaced with sfgfp in order to quantitatively measure the output of the light-sensing system. Replacing this gene with another output is therefore theoretically feasible. Here, the St Andrews iGEM team of 2020 aims to control the expression of the antitoxin ccdA in response to green light. Under activating conditions, expression of ccdA will be maximised whilst also minimising leaky expression from PcpcG2 by using a truncated version of the promoter (PcpcG2-172), characterised by Schmidl et al. (2014).

By regulating expression of ccdA, the dual functionality of the antitoxin can be utilised to prevent ccdB-gyrase formation (where ccdB is previously expressed in BBa_K3634012) and allow for precise transcriptional control of a desired output gene (see BBa_K3634014) (Vandervelde et al., 2017).

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
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]