Difference between revisions of "Part:BBa K2012015"
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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.
 
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.
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<p>Promoter cpcG2 is a 238bp green-light  activated promoter from the genome of Synechocystis PCC6803. We tested the  efficiency of the promoter by measuring the fluorescence of output sfGFP when  bacteria are illuminated with green, red or no light. (For more detail, please  view our wiki: <a href="http://2016.igem.org/Team:HZAU-China/Experiments" target="_blank">http://2016.igem.org/Team:HZAU-China/Experiments</a>)<br/>
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<div style="text-align:left"><img alt="00.1" src="https://static.igem.org/mediawiki/parts/7/71/T--HZAU--China--CcaS-CcaR.jpg" style="float:none;width:400px;height:240px" ><br/>
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</span><strong>Figure 1. Mechanism of CcaS-CcaR system<span>.</span></strong><span><span>&nbsp;<br/>
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<span>&nbsp;</span></span></span></span><span>CcaS is produced in a green-absorbing ground state, termed Pg. Absorption of green light flips CcaS to a kinase-active red-absorbing state (Pr) that phosphorylates the response regulator CcaR, which then binds to the G-box operator within cpcG2 promoter and activates transcription. Absorption of red light switches CcaS Pr back to Pg, which dephosphorylates P-CcaR, deactivating transcription.</span></div>
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<div style="text-align:left"><img alt="00.1" height="293" src="https://static.igem.org/mediawiki/parts/d/db/Hzau-china_cpcg2.jpg" style="float:none" width="480"></div>
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<p><span style="font-weight:bold">Figure 2. Mean fluorescence output in CcaS-CcaR system</span><strong>.</strong></p>
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Revision as of 10:45, 17 October 2016


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.

Promoter cpcG2 is a 238bp green-light activated promoter from the genome of Synechocystis PCC6803. We tested the efficiency of the promoter by measuring the fluorescence of output sfGFP when bacteria are illuminated with green, red or no light. (For more detail, please view our wiki: <a href="http://2016.igem.org/Team:HZAU-China/Experiments" target="_blank">http://2016.igem.org/Team:HZAU-China/Experiments</a>)

<img alt="00.1" src="T--HZAU--China--CcaS-CcaR.jpg" style="float:none;width:400px;height:240px" >

 
Figure 1. Mechanism of CcaS-CcaR system. 

 

 CcaS is produced in a green-absorbing ground state, termed Pg. Absorption of green light flips CcaS to a kinase-active red-absorbing state (Pr) that phosphorylates the response regulator CcaR, which then binds to the G-box operator within cpcG2 promoter and activates transcription. Absorption of red light switches CcaS Pr back to Pg, which dephosphorylates P-CcaR, deactivating transcription.

<img alt="00.1" height="293" src="Hzau-china_cpcg2.jpg" style="float:none" width="480">

Figure 2. Mean fluorescence output in CcaS-CcaR system.

 

 

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.

More importantly, leaked expression in darkness significantly reduced after truncation of the constitutive promoter.

 

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]