Difference between revisions of "Team:FAFU-CHINA/Results"

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<p>Here you can describe the results of your project and your future plans. </p>
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<h5>What should this page contain?</h5>
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<ul>
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<li> Clearly and objectively describe the results of your work.</li>
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<li> Future plans for the project </li>
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<li> Considerations for replicating the experiments </li>
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<h5> Project Achievements </h5>
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
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<li>A list of linked bullet points of the successful results during your project</li>
 
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<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
 
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<h5>Inspiration</h5>
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<p>See how other teams presented their results.</p>
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<ul>
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
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<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
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= <span style="font-family:'Times New Roman'">Result</span> =
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<div><p style="text-indent:12pt"><span style="font-size:12pt;font-family:&quot">&nbsp;&nbsp;This year, FAFU-CHINA team focused toutilize CC530&nbsp;<i>Chlamydomonas reintmrdtii</i>to &nbsp;&nbsp;express&nbsp;<i>Cry</i>&nbsp;and&nbsp;<i>Cyt</i>&nbsp;genes which cloned from&nbsp;<i>BRC-LLP29 Bacillus thuringiensis&nbsp;</i>strainto kill the &nbsp;&nbsp;larvae of mosquitoes.</span></p>
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<p style="text-indent:12pt"><span style="font-size:12pt;font-family:&quot">&nbsp;&nbsp;This project owns various advantagesexpect for the express platform based &nbsp; &nbsp; &nbsp;&nbsp;on&nbsp;<i>Chlamydomonasreintmrdtii.</i>&nbsp;We also improve the toxicity of&nbsp;<i>Cry</i>&nbsp;and&nbsp;<i>Cyt</i>&nbsp;and improveparts as &nbsp;&nbsp;following.</span></p>
 +
<p style="margin-left:27pt;text-indent:-18pt"><i><span style="font-size:12pt;font-family:&quot">1.<span style="font-style:normal;font-variant-numeric:normal;font-stretch:normal;font-size:7pt;font-family:&quot">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></span></i><span style="font-size:12pt;font-family:&quot">Firstly, we decide toexpress&nbsp;<i>Cry4a</i></span><i><span style="font-size:12pt">，</span></i><i><span style="font-size:12pt;font-family:&quot">Cry10a</span></i><i><span style="font-size:12pt">，</span></i><i><span style="font-size:12pt;font-family:&quot">Cry11a</span></i><i><span style="font-size:12pt">，</span></i><i><span style="font-size:12pt;font-family:&quot">Cyt1</span></i><i><span style="font-size:12pt">，</span></i><i><span style="font-size:12pt;font-family:&quot">Cyt2</span></i><span style="font-size:12pt;font-family:&quot">genes which cloned from&nbsp;<i>BRC- &nbsp;LLP29Bacillus thuringiensis&nbsp;</i>strain in&nbsp;<i>E.coli</i>. Due to the homologies of&nbsp;<i>Cry</i>genes, we were stocked &nbsp;in cloning. With the support from ShanghaiTechChina_Bteam, we constructed Cry11a-pET32a (+) and &nbsp;Cyt1-pET32a (+) express vectors. Andtested the expression of&nbsp;<i>Cyt1</i>&nbsp;bySDS-Page.<i></i></span></p>
 +
<p style="margin-left:27pt;text-indent:-18pt"><i><span style="font-size:12pt;font-family:&quot">2.<span style="font-style:normal;font-variant-numeric:normal;font-stretch:normal;font-size:7pt;font-family:&quot">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></span></i><span style="font-size:12pt;font-family:&quot">Firstly, we decide toexpress&nbsp;<i>Cry4a</i></span><i><span style="font-size:12pt">，</span></i><i><span style="font-size:12pt;font-family:&quot">Cry10a</span></i><i><span style="font-size:12pt">，</span></i><i><span style="font-size:12pt;font-family:&quot">Cry11a</span></i><i><span style="font-size:12pt">，</span></i><i><span style="font-size:12pt;font-family:&quot">Cyt1</span></i><i><span style="font-size:12pt">，</span></i><i><span style="font-size:12pt;font-family:&quot">Cyt2</span></i><span style="font-size:12pt;font-family:&quot">genes which cloned from&nbsp;<i>BRC- &nbsp;LLP29Bacillus thuringiensis&nbsp;</i>strain in&nbsp;<i>E.coli</i>.<i></i></span></p>
 +
<p style="margin-left:27pt;text-indent:-18pt"><span style="font-size:12pt;font-family:&quot">3.<span style="font-variant-numeric:normal;font-stretch:normal;font-size:7pt;font-family:&quot">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></span><span style="font-size:12pt;font-family:&quot">Synthesizing<i>Cry</i>&nbsp;and&nbsp;<i>Cyt</i>&nbsp;toxin genes based on&nbsp;<i>Chlamydomonasreintmrdtii</i>&nbsp;codon-optimized &nbsp;to improve the express level.</span></p>
 +
<p style="margin-left:27pt;text-indent:-18pt"><span style="font-size:12pt;font-family:&quot">4.<span style="font-variant-numeric:normal;font-stretch:normal;font-size:7pt;font-family:&quot">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></span><span style="font-size:12pt;font-family:&quot">Toincrease toxicity to the larvae of mosquitoes and the potential possibility ofresistance, &nbsp;we utilized 2A peptide system to co-express&nbsp;<i>Cry</i>&nbsp;and&nbsp;<i>Cyt</i>&nbsp;toxins.</span></p>
 +
<p style="margin-left:27pt;text-indent:-18pt"><span style="font-size:12pt;font-family:&quot">5.<span style="font-variant-numeric:normal;font-stretch:normal;font-size:7pt;font-family:&quot">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></span><span style="font-size:12pt;font-family:&quot">Basedon the different toxicities of&nbsp;<i>Cry</i>and&nbsp;<i>Cyt</i>, we contrasted six toxingroups to select &nbsp;the best group.</span></p>
 +
<p style="margin-left:27pt;text-indent:-18pt"><span style="font-size:12pt;font-family:&quot">6.<span style="font-variant-numeric:normal;font-stretch:normal;font-size:7pt;font-family:&quot">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></span><span style="font-size:12pt;font-family:&quot">Theefficiency of Hsp70A-Rbc S2 promoter can be improved when environmentaltemperature is &nbsp;larger than 40</span><span style="font-size:12pt;font-family:&quot">℃</span><span style="font-size:12pt;font-family:&quot">.It means that the toxins express device will express more toxins in &nbsp;summer.Meanwhile, with the help from NEU-China, we tested the heat shock inducedresult by q-PCR and &nbsp;confocal laser scanning microscope (CLSM).</span></p>
 +
<p style="margin-left:27pt;text-indent:-18pt"><span style="font-size:12pt;font-family:&quot">7.<span style="font-variant-numeric:normal;font-stretch:normal;font-size:7pt;font-family:&quot">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></span><span style="font-size:12pt;font-family:&quot">Basedon the bioinformatics analysis, the result showed that the GC% wassignificantly &nbsp;different between&nbsp;<i>Bacillusthuringiensis</i>&nbsp;and&nbsp;<i>Chlamydomonasreintmrdtii.&nbsp;</i>So with the technology &nbsp;support by Genes for life-DNA Companyin Suzhou, we &nbsp;optimized&nbsp;<i>EGFP</i></span><i><span style="font-size:12pt">，</span></i><i><span style="font-size:12pt;font-family:&quot">Cry4a</span></i><i><span style="font-size:12pt">，</span></i><i><span style="font-size:12pt;font-family:&quot">Cry10a</span></i><i><span style="font-size:12pt">，</span></i><i><span style="font-size:12pt;font-family:&quot">Cry11a</span></i><i><span style="font-size:12pt">，</span></i><i><span style="font-size:12pt;font-family:&quot">Cyt1</span></i><i><span style="font-size:12pt">，</span></i><i><span style="font-size:12pt;font-family:&quot">Cyt2</span></i><span style="font-size:12pt;font-family:&quot">&nbsp;genes based on codonoptimization. We &nbsp;standardized these genes into psb1c3 parts and submitted them.You can find more information by this &nbsp;link:</span></p>
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<p style="margin-left:27pt;text-indent:-18pt"><span style="font-size:12pt;font-family:&quot">8.<span style="font-variant-numeric:normal;font-stretch:normal;font-size:7pt;font-family:&quot">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></span><span style="font-size:12pt;font-family:&quot">Basedon the express vector pChlamy-3 which provide by Huiying Zhang, we constructed13 &nbsp;express vector in&nbsp;<i>Chlamydomonasreintmrdtii.</i></span></p>
 +
<p style="margin-left:27pt;text-indent:-18pt"><span style="font-size:12pt;font-family:&quot">9.<span style="font-variant-numeric:normal;font-stretch:normal;font-size:7pt;font-family:&quot">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></span><i><span style="font-size:12pt;font-family:&quot">Bacillus thuringiensis&nbsp;</span></i><span style="font-size:12pt;font-family:&quot">ishot spot in previous iGEM competition. There are &nbsp;various&nbsp;<i>Cry</i>&nbsp;genes which reserved in Parts stock. Based on the toxin expressplatform in our &nbsp;project, we improve NCTU_Formosa BBa_K332011 parts whichencodes&nbsp;<i>Cry11Aa</i>&nbsp;by codonoptimization. And &nbsp;we submitted the improved part BBa_K2074023.Meanwhile, wealso improved BBa_E0040 part which encodes &nbsp;wild type GFP. And we submittedBBa_K2074026 which encodes eGFP. These improvements can help another &nbsp;teams toutilize&nbsp;<i>Chlamydomonas reintmrdtii.</i></span></p>
 +
<p style="text-indent:12pt"><span style="font-size:12pt;font-family:&quot">&nbsp; &nbsp; We also helped ShanghaiTechChina_B,BNU-China and BNU-China team do experiments and offered a &nbsp; &nbsp; &nbsp; protocol to JFNLS_China. You can find the details in the experiments wiki pages.</span></p>
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Latest revision as of 06:34, 8 November 2016

Contents

Team FAFU-CHINA

Result

  This year, FAFU-CHINA team focused toutilize CC530 Chlamydomonas reintmrdtiito   express Cry and Cyt genes which cloned from BRC-LLP29 Bacillus thuringiensis strainto kill the   larvae of mosquitoes.

  This project owns various advantagesexpect for the express platform based       on Chlamydomonasreintmrdtii. We also improve the toxicity of Cry and Cyt and improveparts as   following.

1.     Firstly, we decide toexpress Cry4a，Cry10a，Cry11a，Cyt1，Cyt2genes which cloned from BRC-  LLP29Bacillus thuringiensis strain in E.coli. Due to the homologies of Crygenes, we were stocked  in cloning. With the support from ShanghaiTechChina_Bteam, we constructed Cry11a-pET32a (+) and  Cyt1-pET32a (+) express vectors. Andtested the expression of Cyt1 bySDS-Page.

2.     Firstly, we decide toexpress Cry4a，Cry10a，Cry11a，Cyt1，Cyt2genes which cloned from BRC-  LLP29Bacillus thuringiensis strain in E.coli.

3.      SynthesizingCry and Cyt toxin genes based on Chlamydomonasreintmrdtii codon-optimized  to improve the express level.

4.      Toincrease toxicity to the larvae of mosquitoes and the potential possibility ofresistance,  we utilized 2A peptide system to co-express Cry and Cyt toxins.

5.      Basedon the different toxicities of Cryand Cyt, we contrasted six toxingroups to select  the best group.

6.      Theefficiency of Hsp70A-Rbc S2 promoter can be improved when environmentaltemperature is  larger than 40℃.It means that the toxins express device will express more toxins in  summer.Meanwhile, with the help from NEU-China, we tested the heat shock inducedresult by q-PCR and  confocal laser scanning microscope (CLSM).

7.      Basedon the bioinformatics analysis, the result showed that the GC% wassignificantly  different between Bacillusthuringiensis and Chlamydomonasreintmrdtii. So with the technology  support by Genes for life-DNA Companyin Suzhou, we  optimized EGFP，Cry4a，Cry10a，Cry11a，Cyt1，Cyt2 genes based on codonoptimization. We  standardized these genes into psb1c3 parts and submitted them.You can find more information by this  link:

8.      Basedon the express vector pChlamy-3 which provide by Huiying Zhang, we constructed13  express vector in Chlamydomonasreintmrdtii.

9.      Bacillus thuringiensis ishot spot in previous iGEM competition. There are  various Cry genes which reserved in Parts stock. Based on the toxin expressplatform in our  project, we improve NCTU_Formosa BBa_K332011 parts whichencodes Cry11Aa by codonoptimization. And  we submitted the improved part BBa_K2074023.Meanwhile, wealso improved BBa_E0040 part which encodes  wild type GFP. And we submittedBBa_K2074026 which encodes eGFP. These improvements can help another  teams toutilize Chlamydomonas reintmrdtii.

    We also helped ShanghaiTechChina_B,BNU-China and BNU-China team do experiments and offered a       protocol to JFNLS_China. You can find the details in the experiments wiki pages.