Team:XHD-Wuhan-B-China/Contribution
Contribution
Measurement of CcaS-CcaR system under new circumstances
See BBa_K592002 : CcaR
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 this modified two-component system composed of CcaS#10 and a series of CcaRs with different transcription intensities.
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 this 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 CcaS-CcaR system 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 CcaS-CcaR system 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.
References
[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.
[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.