Difference between revisions of "Part:BBa K3634006"

Revision as of 12:56, 5 August 2020

ccaS (Codon 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) 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, 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 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. When varying green light (520nm) intensity from 0.00 to 2.86W/m^2 in the presence of 1.05W/m^2 red light (650nm), Schmidl et al. (2014) produced a close to maximum response in comparison to no red light incident. As the average irradiance of red light per nm from the sun spectra at solar noon is comparable to that used here, we propose the system can be activated with close to maximum efficiency in broad daylight before inactivation when returned to dark conditions. Using the specified circuitry from Schmidl et al. (2014), the systems output under activating conditions will be maximised whilst also minimising leaky expression from PcpcG2.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 2078
Illegal BamHI site found at 1417
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1449
1000
COMPATIBLE WITH RFC[1000]

@@ Line 5: / Line 5: @@
 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, 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.
+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.