Coding

Part:BBa_K592002

Designed by: Erik Lundin   Group: iGEM11_Uppsala-Sweden   (2011-09-09)
Revision as of 15:05, 9 August 2020 by Ls268 (Talk | contribs)

ccaR

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.

St Andrews iGEM 2020 carried out further codon optimisation for E.coli for the whole BBa_K592002 sequence using the IDT codon optimisation tool. Optimisation created no new illegal restriction sites allowing the part (BBa_K3634017) to be used at RFC[10] and RFC[1000] standard with codon optimisation.


Figure 1: Plates containing the pSR58.6 (left) and pNO286-3 (right) plasmids under UV light. The pSR58.6 plasmid contains CcaR and superfolder GFP. The pNO286-3 plasmid contains the mini-CcaS gene, along with ho1 and pcyA.



Figure 2: Plate containing cells co-transformed with both pSR58.6 and pNO286-3 plasmids.


Figure 3: Light sensor readings from plates containing optogenetic plasmids






























































Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 402
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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