Part:BBa_K592002

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.

BBa_K3634017

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]

iGEM 2021 XHD-Wuhan-B-China, new documentation (For Bronze)

Summary: Measurement of CcaS-CcaR system under new circumstances

PcpcG2-172 is a 172bp green-light induced promoter constructed by Sebastian R. Schmidl group [1].CcaS phosphorylates CcaR under green light and induces expression of cpcG2 [2].A study of Mitsuharu Nakajima group reveals that CcaS#10 works well when two PAS domains were deleted from the original sequence [3].Therefore, we tested the modified two-component system composed of CcaS#10 and a series of CcaRs with different transcription intensities.

Figure1. Light inducible CcaS-CcaR two component system. (A) Structure of the CcaS-CcaR circuit. (B) Measurement of PcpcG2-172 under red light (660nm) or green light (520nm). The experiment was carried out in MG1655-△EnvZ strain, using a 24-wall plate adapted LED device. Graphic shows that among four tested promoters, J23117-CcaR gives PcpcG2-172 best dynamic range of 8.3-fold change. When using stronger promoters J23114 and J23115, this dynamic range narrows as leakage under red light getting larger.

Figure2. Light-control devices used in this experiment.

Reference

[1] Schmidl SR, Sheth RU, Wu A, Tabor JJ. Refactoring and optimization of light-switchable Escherichia coli two-component systems. ACS Synth Biol. 2014 Nov 21;3(11):820-31.
[2] Hirose Y, Shimada T, Narikawa R, Katayama M, Ikeuchi M. Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. Proc Natl Acad Sci U S A. 2008 Jul 15;105(28):9528-33.
[3] Nakajima M, Ferri S, Rögner M, Sode K. Construction of a Miniaturized Chromatic Acclimation Sensor from Cyanobacteria with Reversed Response to a Light Signal. Sci Rep. 2016 Nov 24;6:37595.