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.
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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.(For more detail, please  view our wiki: "http://2016.igem.org/Team:HZAU-China/Experiments" )
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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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<div style="margin-left:auto;margin-right:auto;position:relative"><p style="text-align:justify;text-justify:inter-ideograph"><span style="font-family:Calibri"><img height="318.92999267578125" src="https://static.igem.org/mediawiki/2016/5/5f/Eeeee.jpeg" width="480.0"></span><span style="font-family:Calibri"><span></span></span></p>
 
<div style="margin-left:auto;margin-right:auto;position:relative"><p style="text-align:justify;text-justify:inter-ideograph"><span style="font-family:Calibri"><img height="318.92999267578125" src="https://static.igem.org/mediawiki/2016/5/5f/Eeeee.jpeg" width="480.0"></span><span style="font-family:Calibri"><span></span></span></p>
 
<p style="text-align:justify;text-justify:inter-ideograph"><span style="font-family:Calibri">&nbsp;</span></p>
 
<p style="text-align:justify;text-justify:inter-ideograph"><span style="font-family:Calibri">&nbsp;</span></p>
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<p style="text-align:justify;text-justify:inter-ideograph"><span style="font-family:Calibri">Fluorescence assay of CcaS-CcaR system with PcpcG2-172 (BBa_K2012015) in CL1 (△EnvZ), and PCB (△CcaS) as </span><span style="font-family:Calibri">chromophore</span><span style="font-family:Calibri">. </span><span style="font-family:Calibri"><span></span></span></p>
 
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<p style="text-align:justify;text-justify:inter-ideograph"><span style="font-family:Calibri">F</span><span style="font-family:Calibri">luorescence under green light is 1.81-folds of red light, proving that green light activates output expression, the device works well. </span><span style="font-family:Calibri">PcpcG2-172 </span><span style="font-family:Calibri">shows high efficiency.<span></span></span></p>
 
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<p style="text-align:justify;text-justify:inter-ideograph"><span style="font-family:Calibri">More importantly, l</span><span style="font-family:Calibri">eaked expression in darkness significantly reduced after truncation of the constitutive promoter. </span><span style="font-family:Calibri;font-weight:bold"><span></span></span></p>
 
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<p style="text-align:justify;text-justify:inter-ideograph"><span style="font-family:Calibri"><strong>Figure 1.</strong> Fluorescence assay of CcaS-CcaR system with PcpcG2-172 (BBa_K2012015) in CL1 (△EnvZ), and PCB (△CcaS) as </span><span style="font-family:Calibri">chromophore</span><span style="font-family:Calibri">. </span><span style="font-family:Calibri"><span></span></span></p>
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<p style="text-align:justify;text-justify:inter-ideograph"><span style="font-family:Calibri">F</span><span style="font-family:Calibri">luorescence under green light is 1.81-folds of red light, proving that green light activates output expression, the device works well. </span><span style="font-family:Calibri">PcpcG2-172 </span><span style="font-family:Calibri">shows high efficiency.<span></span></span></p>
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<b>Improvement</b><br>A truncated promoter PcpcG2-68 has the similar pattern under the control of green/red light as PcpcG2-172, but is shorter and more convenient to construct. For example, when constructing an array of sgRNAs, PcpcG2-68 has a big advantage over PcpcG2-172: reducing the volume of the circuit and cheaper to synthesis.<br> [[File:T--XHD-Wuhan-B-China--111(1).png|900px|thumb|left|]]<br>Please view https://parts.igem.org/Part:BBa_K3921000 for more details.
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2012015 parameters</partinfo>
 
<partinfo>BBa_K2012015 parameters</partinfo>
 
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<span id="IGEM 2021 XHD- Wuhan-B-China"><br><br><br><br><br></span>

Latest revision as of 06:41, 21 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.




Improvement
A truncated promoter PcpcG2-68 has the similar pattern under the control of green/red light as PcpcG2-172, but is shorter and more convenient to construct. For example, when constructing an array of sgRNAs, PcpcG2-68 has a big advantage over PcpcG2-172: reducing the volume of the circuit and cheaper to synthesis.
T--XHD-Wuhan-B-China--111(1).png

Please view https://parts.igem.org/Part:BBa_K3921000 for more details.

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]