Team:UNITN-Trento/Project/Datapage

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<img src="https://static.igem.org/mediawiki/2013/5/57/Tn-2013-datapage-Path.jpg" style="max-width:950px; margin:2em auto; box-shadow: 2px 2px 4px #323232;" />
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We envision this genetic circuit in which production of ethylene and methyl-salycilate (MeSA) are regulated by light. In a dark state, the sensor protein YF-1 auto-phosphorilates itself, dimerize, and transfer the phosphate to the response regulator element FixJ. FixJ is so activated and is able to bind pFixK2 promoter, activating transcription of cI (inhibitor protein) that ultimately blocks EFE production. At the same time, it is transcribed Bmst1, which regulates MeSA synthesis thus slowing down fruit ripening. In a blue-light state the sensor protein YF-1 does not phosphorilate itself, so pFixK2 promoter is in the off state: ethylene is produced thus promoting ripening while MeSA production is blocked.  
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We envision the construction of a genetic circuit that either produces ethylene or methyl-salicylate (MeSA) in response to either the presence or absence of light. In the dark state the sensor protein YF-1 first auto-phosphorylates and then phosphorylates the response regulator FixJ. Phosphorylated FixJ is then able to bind to the pFixK2 promoter thereby activating transcription of cI (the inhibitor protein) that ultimately blocks EFE production. At the same time, phosphorylated FixJ activates the transcription of Bsmt1, which initiates the synthesis of MeSA, thus slowing down fruit ripening. <br/><br/>
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In the presence of blue-light, the sensor protein YF-1 does not auto-phosphorylate and does not phosphorylate FixJ. The RNA polymerase does not recognize the pFixK2 promoter. The result is that ethylene is produced and MeSA synthesis is blocked. In other words, fruit ripening is induced.
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These are our best characterized parts on the system
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<span class="tn-subtitle">These are our best characterized parts on the system</span>
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<span class="tn-sub-subtitle"><b>Best natural part</b></span>
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<a href="http://parts.igem.org/Part:BBa_K1065002">BBa_K1065002</a>, 2-Oxoglutarate Oxygenase/Decarboxylase Ethylene Forming Enzyme (EFE). This part was well characterized under the control of different inducible promoters in both <i>E. coli</i> (NEB10beta cells) and in <i>B. subtilis</i> (str.168).
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<span class="tn-sub-subtitle"><b>Best engineered device</b><br></span>
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<a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a>, blue light regulated amilCP producing device. This device includes an inverter cassette (composed by cI protein and pLambda promoter) that ultimately allows production of the chromoprotein amilCP only when the culture is exposed to light.
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<span class="tn-sub-subtitle"><b>Best improved part</b><br></span>
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<a href="http://parts.igem.org/Part:BBa_K1065305">BBa_K1065305</a>, blue light regulated amilGFP producing device (a yellow fluorescent protein). The part <a href="http://parts.igem.org/Part:BBa_K952003">BBa_K952003</a>, extracted from the registry, was not working because it was missing the RBS between pFixK2 promoter and amilGFP coding sequence. We improved this part by mutagenesis and demonstrated that it is able to produce amilGFP in the dark. .<br/><br/>
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<span class="subtitle">We also characterized the following parts:</span>
<ul>
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<li><b>Best natural part:</b></li> <a href="http://parts.igem.org/Part:BBa_K1065000">BBa_K1065000</a>, 2-Oxoglutarate Oxygenase/Decarboxylase Ethylene Forming Enzyme (EFE). This part was well characterized under the control of different inducible promoters in both <i>E. coli</i> (NEB10beta cells) and in <i>B. subtilis</i> (str.168).
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<li>
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<li><b>Best engineered device:</b> <a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a>, blue light regulated AmylCP producing device. This device includes an inverter cassette (composed by cI protein and pLambda promoter) that ultimately allows production of the chromoprotein AmilCP only when the culture is exposed to light.</li>
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<a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a>: methyl-salycilate producing device.
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<li><b>Best improved part:</b> <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>, blue light regulated AmilGFP producing device (a yellow fluorescent protein). The part <a href="http://parts.igem.org/Part:BBa_K952003">BBa_K952003</a>, extracted from the registry, was not working because it was missing the RBS between pFixK2 promoter and AmilGFP coding sequence. We demonstrated than that our part (created by mutagenesis) produced successfully AmilGFP when the culture was mantained in the dark.</li>
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</li>
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<li>
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<a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a>: an improved version of our methyl-salycilate producing device. This device include the SAM synthetase gene to boost wintergreen production.
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</li>
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<li>
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<a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>: EFE under the control of the blue light circuit with inverter. This device produces ethylene upon photoinduction.
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</li>
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                        <li>
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<a href="http://parts.igem.org/Part:BBa_K1065309">BBa_K1065309</a>: EFE under the control of the blue light circuit without inverter. This device successfully produced ethylene in the dark.
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</li>
</ul>
</ul>
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<span class="tn-effect-link"><a href="https://2013.igem.org/Team:UNITN-Trento/Project/Fruit_ripening">Check how we exploited our parts to ripen fruit!</a></span>
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    <span>This is the end of the tour!<br />Thank you for your company!</span>
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Latest revision as of 13:35, 28 October 2013

We envision the construction of a genetic circuit that either produces ethylene or methyl-salicylate (MeSA) in response to either the presence or absence of light. In the dark state the sensor protein YF-1 first auto-phosphorylates and then phosphorylates the response regulator FixJ. Phosphorylated FixJ is then able to bind to the pFixK2 promoter thereby activating transcription of cI (the inhibitor protein) that ultimately blocks EFE production. At the same time, phosphorylated FixJ activates the transcription of Bsmt1, which initiates the synthesis of MeSA, thus slowing down fruit ripening.

In the presence of blue-light, the sensor protein YF-1 does not auto-phosphorylate and does not phosphorylate FixJ. The RNA polymerase does not recognize the pFixK2 promoter. The result is that ethylene is produced and MeSA synthesis is blocked. In other words, fruit ripening is induced.
These are our best characterized parts on the system Best natural part BBa_K1065002, 2-Oxoglutarate Oxygenase/Decarboxylase Ethylene Forming Enzyme (EFE). This part was well characterized under the control of different inducible promoters in both E. coli (NEB10beta cells) and in B. subtilis (str.168). Best engineered device
 BBa_K1065310, blue light regulated amilCP producing device. This device includes an inverter cassette (composed by cI protein and pLambda promoter) that ultimately allows production of the chromoprotein amilCP only when the culture is exposed to light. Best improved part
 BBa_K1065305, blue light regulated amilGFP producing device (a yellow fluorescent protein). The part BBa_K952003, extracted from the registry, was not working because it was missing the RBS between pFixK2 promoter and amilGFP coding sequence. We improved this part by mutagenesis and demonstrated that it is able to produce amilGFP in the dark. .

We also characterized the following parts:
  • BBa_K1065102: methyl-salycilate producing device.
  • BBa_K1065106: an improved version of our methyl-salycilate producing device. This device include the SAM synthetase gene to boost wintergreen production.
  • BBa_K1065311: EFE under the control of the blue light circuit with inverter. This device produces ethylene upon photoinduction.
  • BBa_K1065309: EFE under the control of the blue light circuit without inverter. This device successfully produced ethylene in the dark.
Check how we exploited our parts to ripen fruit!
This is the end of the tour!
Thank you for your company!
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