Coding

Part:BBa_K592001

Designed by: Lei Sun   Group: iGEM11_Uppsala-Sweden   (2011-09-21)

CcaS, green light sensor from Synechocystis sp. PCC6803

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) 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). CcaS, alongside the response regulator ccaR 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. PCB is made by ho1 and PcyA.

A new part (BBa_K2627000) constructed by SDU-China was improved from CcaS by deleting about 800 bases corresponding to two PAS domains since we were inspired by Professor Koji’s work. Part BBa_K2627000 (CcaS#4) is a varient of CcaS having an opposite response to green/red light. High intensity of fluorescence can be seen when bacteria are cultured under red light while fluorescence is pretty weak under green light.

St Andrews iGEM 2020 carried out further codon optimisation for E.coli for the whole BBa_K592001 sequence using the IDT codon optimisation tool. Optimisation created 3xEcoRI, 1xPstI and 1xSapI illegal restriction sites which were then removed by in silico point mutagenesis. This allowed for the part (BBa_K3634006) to be used at RFC[10] and RFC[1000] standard with codon optimisation.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 606
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 2252
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1270
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None