Difference between revisions of "Team:WHU-China/Improve"

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{{WHU-China}}
 
{{WHU-China}}
 
<html>
 
<html>
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<meta http-equiv="Content-Type" content="text/html; charset=utf-8" />
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<title>Safety</title>
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  overflow-y:auto;
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h1,h2,h3,h4{
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  font-family:"Microsoft YaHei","Segoe UI", "Lucida Grande", Helvetica, Arial,sans-serif;
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}
  
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<div class="column full_size judges-will-not-evaluate">
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img{
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<h3>★  ALERT! </h3>
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width:100%;
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<p>This page is used by the judges to evaluate your team for the <a href="https://2018.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2018.igem.org/Judging/Awards"> award listed below</a>. </p>
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2018.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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</head>
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<body>
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<header>
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<div class="container agile-banner_nav">
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<div class="row header-top">
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                  <div class="col-md-5 top-left p-0">
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<p> IGEM-WHU </p>
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</div>
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<div class="col-md-7 top-right p-0">
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</div>
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                </div>
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<nav class="navbar navbar-expand-lg navbar-light bg-light">
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<h1><a class="navbar-brand" href="https://2018.igem.org/Team:WHU-China">IGEM - WHU</a></h1>
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<a href="#" class="dropdown-toggle nav-link" data-toggle="dropdown">Project
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<b class="caret"></b>
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<ul class="dropdown-menu agile_short_dropdown">
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<li><a href="https://2018.igem.org/Team:WHU-China/Description">Description</a></li>
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<li><a href="https://2018.igem.org/Team:WHU-China/Design">Design</a></li>
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<li><a href="https://2018.igem.org/Team:WHU-China/Experiments">Experiments</a></li>
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                                <li><a href="https://2018.igem.org/Team:WHU-China/Notebook">Notebook</a></li>
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                                <li><a href="https://2018.igem.org/Team:WHU-China/Protocol">Protocol</a></li>
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<li><a href="https://2018.igem.org/Team:WHU-China/Model">Modelling</a></li>
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<li><a href="https://2018.igem.org/Team:WHU-China/Demonstrate">Demonstrate</a></li>
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<li><a href="https://2018.igem.org/Team:WHU-China/Improve">Improve</a></li>
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<li><a href="https://2018.igem.org/Team:WHU-China/Future_plan">Furture plans</a></li>
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<li><a href="https://2018.igem.org/Team:WHU-China/InterLab">InterLab</a></li>
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                                <li><a href="https://2018.igem.org/Team:WHU-China/Safety">Safety</a></li>
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</ul>
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                        <li><a href="https://2018.igem.org/Team:WHU-China/Team">Team members</a></li>
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                        <li><a href="https://2018.igem.org/Team:WHU-China/Parts">Overview</a></li>
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                        <li><a href="https://2018.igem.org/Team:WHU-China/Parts/Basic_parts">Basic parts</a></li>
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                                        <li><a href="https://2018.igem.org/Team:WHU-China/Parts/Composite parts">Composite parts</a></li>
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    <li><a href="https://2018.igem.org/Team:WHU-China/Human_Practices">Human Practice</a></li>
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                      <li><a href="https://2018.igem.org/Team:WHU-China/Public_Engagement">Education engagement</a></li>
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<li class="dropdown nav-item">
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<a href="#" class="dropdown-toggle nav-link" data-toggle="dropdown">Awards
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<li><a href="https://2018.igem.org/Team:WHU-China/Medal_criteria">Meadal criteria</a></li>
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                <li><a href="https://2018.igem.org/Team:WHU-China/Hardware">Hardware</a></li>
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                <li><a href="https://2018.igem.org/Team:WHU-China/Measurement">Measurement</a></li>
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</ul>
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<li class="nav-item pr-lg-0">
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<a class="nav-link pr-lg-0" href="https://2018.igem.org/Team:WHU-China/Device">Device</a>
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</ul>
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</div>
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</nav>
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</div>
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</header>
  
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<div class="column full_size">
 
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<h1>Improve</h1>
 
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<p>For teams seeking to improve upon a previous part or project, you should document all of your work on this page. Please remember to include all part measurement and characterization data on the part page on the Registry. Please include a link to your improved part on this page.</p>
 
  
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<h3>Gold Medal Criterion #2</h3>
 
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<p><b>Standard Tracks:</b> Create a new part that has a functional improvement upon an existing BioBrick part. The sequences of the new and existing parts must be different. You must perform experiments with both parts to demonstrate this improvement.  Document the experimental characterization on the Part's Main Page on the Registry for both the existing and new parts. Both the new and existing Main Page of each Part’s Registry entry must reference each other. Submit a sample of the new part to the Registry.
 
  
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The existing part must NOT be from your 2018 part number range and must be different from the part documented in bronze #4.
 
  
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<br><br>
 
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<b>Special Tracks:</b> Improve the function of an existing iGEM project (that your current team did not originally create) and display your achievement on your wiki.</p>
 
  
  
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</div>
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<div class="c_row">
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<br/><br/><br/><br/><br/><br/>
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<h1>Improve</h1>
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<h2>BBa_K2789001 over BBa_K608102</h2><br/>
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<p style="font-size:24px;">This year we have improved the part BBa_K608102 designed by the 11-Freiburg iGEM team. This is a light control component Ccas. We want to use this component, but there are illegal EcoRI and SpeI sites in the sequence that do not comply with the RFC10 standard.  Since this component needs to be used with another part Ccar, there will be problems during the assembly process. This team also encountered this problem, and this part has not been characterized.<br/><br/>
  
 +
In order to solve this problem, we re-synthesized the entire sequence (including Ccas and Ccar), optimized the codons, eliminating all the restriction sites that do not meet the RFC10 standard, and assembled our improved pathway BBB_K2789001 Subsequently, we used the promoter PcpcG+GFP, which is regulated by our part to characterize it. <br/><br/>
  
 +
Finally, we proved that Ccas can work in this pathway—it can be activated by green light, thus phosphorylate the Ccar and then turn on the downstream expression of the PcpcG promoter. This process can be ceased by red light as stated in the literature.<br/><br/>
  
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BBa_K608102（2262bp）—（codon optimized to avoid the restriction site that involved in RFC10 standard）—BBa_K2789009(2262bp)
 +
—（add Ccar to make it a complete functional unit）—BBa_K2789001(3416bp,submitted)<br/><br/>
 +
</p>
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<h2>Characterization:</h2>
 +
<br/>
 +
<h3>Experiments:</h3>
 +
<p style="font-size:24px;">
 +
1. Transform the plasmids we have constructed into E.coli BL21 and select the single colony to cultivate in 10ml LB medium overnight.<br/>
 +
2. Transfer 1ml medium after cultivating overnight into the 9ml sterilization LB medium <br/>
 +
3. Cultivate the medium for 2 hours in 37℃<br/>
 +
4. Add the IPTG to activate the gene expression<br/>
 +
5. Get samples of medium after cultivating in 0h, 2.5h and 5h . Totally 3 samples each plasmid. Measure the fluorescence intensity of these samples ( activated with 480 nm light and measure in 530nm light)<br/>
 +
6. After induced for 5 hours, cultivating the transformed E.coli under green light, red light and darkness for 2 hours.<br/>
 +
7. Get samples after activated by different lights and then transform the green light into red light, vice versa , but bacteria in darkness keep dim,
 +
8. Get samples after transforming the light for 1 hour.<br/>
 +
</p>
 +
<h3>Results:</h3>
 +
<div style="margin:0 auto;width:800px;">
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<img src="https://static.igem.org/mediawiki/2018/5/5a/T--WHU-China--wiki-Improve_main1.png"><br/><br/>
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<img src="https://static.igem.org/mediawiki/2018/1/1f/T--WHU-China--wiki-Improve_main2.png"><br/><br/>
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<img src="https://static.igem.org/mediawiki/2018/f/f4/T--WHU-China--wiki-Improve_main3.png"><br/><br/><br/>
 +
</div>
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<p style="font-size:24px;">We transformed the improved part BBa_K2789001 and use PcpcG promoter + GFP, to test the whole light control system, you can see from the picture that after expressing our part for 5h, it can express GFP after exposed to green light. And this process can be ceased after exposed to red light.</p>
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</div>
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<div>
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  <br />
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  <br />
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Latest revision as of 03:25, 18 October 2018

Safety

IGEM-WHU







Improve

BBa_K2789001 over BBa_K608102


This year we have improved the part BBa_K608102 designed by the 11-Freiburg iGEM team. This is a light control component Ccas. We want to use this component, but there are illegal EcoRI and SpeI sites in the sequence that do not comply with the RFC10 standard. Since this component needs to be used with another part Ccar, there will be problems during the assembly process. This team also encountered this problem, and this part has not been characterized.

In order to solve this problem, we re-synthesized the entire sequence (including Ccas and Ccar), optimized the codons, eliminating all the restriction sites that do not meet the RFC10 standard, and assembled our improved pathway BBB_K2789001 Subsequently, we used the promoter PcpcG+GFP, which is regulated by our part to characterize it.

Finally, we proved that Ccas can work in this pathway—it can be activated by green light, thus phosphorylate the Ccar and then turn on the downstream expression of the PcpcG promoter. This process can be ceased by red light as stated in the literature.

BBa_K608102（2262bp）—（codon optimized to avoid the restriction site that involved in RFC10 standard）—BBa_K2789009(2262bp) —（add Ccar to make it a complete functional unit）—BBa_K2789001(3416bp,submitted)

Characterization:


Experiments:

1. Transform the plasmids we have constructed into E.coli BL21 and select the single colony to cultivate in 10ml LB medium overnight.
2. Transfer 1ml medium after cultivating overnight into the 9ml sterilization LB medium
3. Cultivate the medium for 2 hours in 37℃
4. Add the IPTG to activate the gene expression
5. Get samples of medium after cultivating in 0h, 2.5h and 5h . Totally 3 samples each plasmid. Measure the fluorescence intensity of these samples ( activated with 480 nm light and measure in 530nm light)
6. After induced for 5 hours, cultivating the transformed E.coli under green light, red light and darkness for 2 hours.
7. Get samples after activated by different lights and then transform the green light into red light, vice versa , but bacteria in darkness keep dim, 8. Get samples after transforming the light for 1 hour.

Results:








We transformed the improved part BBa_K2789001 and use PcpcG promoter + GFP, to test the whole light control system, you can see from the picture that after expressing our part for 5h, it can express GFP after exposed to green light. And this process can be ceased after exposed to red light.