Difference between revisions of "Part:BBa K592001"

Revision as of 14:46, 9 August 2020

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, 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.

CcaS is a green light-induced sensor protein from the genome of Synechocystis sp. PCC6803. It requires PCB as the chromophore to function. PCB is made by ho1 and PcyA. Furthermore, CcaS works in conjunction with CcaR, its response regulator. This is the green light sensor used by Tabor in his multichromatic light-sensing system which composed of CcaS/CcaR and Cph8/EnvZ.

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 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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 __NOTOC__
 <partinfo>BBa_K592001 short</partinfo>
+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.
 CcaS is a green light-induced sensor protein from the genome of Synechocystis sp. PCC6803. It requires PCB as the chromophore to function. PCB is made by [[Part:BBa_I15008|ho1]] and [[Part:BBa_I15009|PcyA]]. Furthermore, CcaS works in conjunction with [[Part:BBa_K592002|CcaR]], its response regulator. This is the green light sensor used by Tabor in his multichromatic light-sensing system which composed of CcaS/CcaR and Cph8/EnvZ. <br>
 <p>A new part (<partinfo>BBa_K2627000</partinfo>) 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.</p>
+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 to be used at RFC[10] and RFC[1000] standard with codon optimisation.
 <!-- Add more about the biology of this part here