==iGEM 2021 XHD-Wuhan-B-China, new documentation (For Bronze)==
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<h3><b>Group: XHD-Wuhan-B-China</b></h3>
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<h3><b>Author: Kening Chen</b></h3>
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<h3><b>Summary: Measurement of PcpcG2-172 under new circumstances</b></h3>
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<p> 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 PcpcG2-172 in a modified two-component system composed of CcaS#10 and a series of CcaRs with different transcription intensities.</p>
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[[File:T—HZAU-China—Figure1.png|600px|thumb|left|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.]]
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[[File:T—HZAU-China—Figure2.png|600px|thumb|left|Figure2. Light-control devices used in this experiment.]]
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<h3 style="clear:left;">Reference</h3>
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<p>[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.<br>
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[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.<br>
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[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.</p>
Revision as of 05:13, 4 October 2021
PcpcG2-172, a modified PcpcG2 promoter
PcpcG2 promoter is a 238bp green-light activated promoter in Synechocystis PCC 6803(Part:BBa_K592003). The full length promoter is comprised of a G-box region, a CcaR-P activated promoter, and a constitutive promoter, which contributes to the leakiness under red light and low dynamic range. Therefore, we constructed PcpcG2-172, a 172bp truncated cpcG2 promoter deleted for the constitutive promoter.(For more detail, please view our wiki: "http://2016.igem.org/Team:HZAU-China/Experiments" )
Figure 1. Fluorescence assay of CcaS-CcaR system with PcpcG2-172 (BBa_K2012015) in CL1 (△EnvZ), and PCB (△CcaS) as chromophore.
Fluorescence under green light is 1.81-folds of red light, proving that green light activates output expression, the device works well. PcpcG2-172 shows high efficiency.
