Difference between revisions of "Team:ECUST China/Notebook"

Line 41: Line 41:
 
         <p>We started to plan our wet lab arrangement and construction work.</p>
 
         <p>We started to plan our wet lab arrangement and construction work.</p>
 
         <h2>—July 2019</h2>
 
         <h2>—July 2019</h2>
 +
        <p>28th, July
 +
—We went to ShenZhen to participate CCiC 2019.
 
         <p>Our experiment didn’t start until this month, but we quickly assigned the tasks into several team member. In detail, shows as follows:</p>
 
         <p>Our experiment didn’t start until this month, but we quickly assigned the tasks into several team member. In detail, shows as follows:</p>
 
         <ul class="circle">
 
         <ul class="circle">
 
         <li><b>Cellulose degrading ability</b>
 
         <li><b>Cellulose degrading ability</b>
 
         <p>This part was mostly undertaken by Hushijia, and  later assisted by Xianglan. The results could be seen at <a href="https://2019.igem.org/Team:ECUST_China/Results" target="_blank" style="text-decoration: underline;"> here<img style="height: 20px;" src="https://static.igem.org/mediawiki/2019/b/bd/T--ECUST_China--icon_cursor.png"></a>. </p>
 
         <p>This part was mostly undertaken by Hushijia, and  later assisted by Xianglan. The results could be seen at <a href="https://2019.igem.org/Team:ECUST_China/Results" target="_blank" style="text-decoration: underline;"> here<img style="height: 20px;" src="https://static.igem.org/mediawiki/2019/b/bd/T--ECUST_China--icon_cursor.png"></a>. </p>
−
         <p>We firstly attempted to construct the cex and cenA on the pET-28b separately, since the dual inverter plasmid has not been constructed and the pET-28b is a reliable plasmid to express heterologous protein.</p>
+
         <p>2nd, July
−
         <p>At the beginning, we got desired parts from kit by PCR very easily. And through Gibson-assembly method, we ligated the parts we purified. When we tested the ligation section by test primer and first generation sequencing, nonetheless,  there were not a single clone  comprise all the fragments we expected to be ligated.(More details please click the botton below)</p>
+
—We firstly attempted to construct the cex and cenA on the pET-28b separately, since the dual inverter plasmid has not been constructed and the pET-28b is a reliable plasmid to express heterologous protein.</p>
 +
         <p>9th, July
 +
—At the beginning, we got desired parts from kit by PCR very easily. And through Gibson-assembly method, we ligated the parts we purified.
 +
        <p>20th,July
 +
—When we tested the ligation section by test primer and first generation sequencing, nonetheless,  there were not a single clone  comprise all the fragments we expected to be ligated.
 
         <div class="btn"><a href="https://static.igem.org/mediawiki/2019/8/8d/T--ECUST_China--experiments_protocols.pdf" style="padding-left:16px;"> <b style="color: white;">Protocols</b></a></div>   
 
         <div class="btn"><a href="https://static.igem.org/mediawiki/2019/8/8d/T--ECUST_China--experiments_protocols.pdf" style="padding-left:16px;"> <b style="color: white;">Protocols</b></a></div>   
 
         </li>
 
         </li>
Line 53: Line 59:
 
         <p><b>Cellulose producing ability</b></p>
 
         <p><b>Cellulose producing ability</b></p>
 
         <p>This part was firstly undertaken by Xianglan, and then taken over by Jinqianwei and  Miaozongjie. The results could be seen at<a href="https://2019.igem.org/Team:ECUST_China/Results" target="_blank" style="text-decoration: underline;"> here<img style="height: 20px;" src="https://static.igem.org/mediawiki/2019/b/bd/T--ECUST_China--icon_cursor.png"></a>.</p>
 
         <p>This part was firstly undertaken by Xianglan, and then taken over by Jinqianwei and  Miaozongjie. The results could be seen at<a href="https://2019.igem.org/Team:ECUST_China/Results" target="_blank" style="text-decoration: underline;"> here<img style="height: 20px;" src="https://static.igem.org/mediawiki/2019/b/bd/T--ECUST_China--icon_cursor.png"></a>.</p>
−
           <p>We tried to purchase Gluconacetobacter xylinus from</p>
+
           <p>1nd, July
 +
—We tried to purchase Gluconacetobacter xylinus from China General Microbiological Culture Collection Center, but we decided not to finish this order since we didn’t have the protocols and available plasmid in our school, making it almost impossible for us to set it as our chasis.  </p>
 +
        <p>7th, July
 +
—We chose E.coli DH5alpha as our chasis.
 +
        <p>18th,July
 +
—We met some problems about acquiring the acsAB and acsCD from the kit, since they were both about 5500bp.
 +
        <p>26th,July
 +
—Even though we occasionally got the desired fragments, problems still exist in the ligation procedure. We couldn’t ligate the acsAB, and acsCD with neither pET-28b backbone nor pSB1C3 by Gibson-assembly and T4-ligation respectively.
 
         </li>
 
         </li>
 
         <li>
 
         <li>
 
         <p><b>Inversion-control ability</b></p>
 
         <p><b>Inversion-control ability</b></p>
 
         <p>This part was mostly undertaken by Jinqianwei and  Miaozongjie. The results could be seen at <a href="https://2019.igem.org/Team:ECUST_China/Results" target="_blank" style="text-decoration: underline;">here<img style="height: 20px;" src="https://static.igem.org/mediawiki/2019/b/bd/T--ECUST_China--icon_cursor.png"></a>.</p>
 
         <p>This part was mostly undertaken by Jinqianwei and  Miaozongjie. The results could be seen at <a href="https://2019.igem.org/Team:ECUST_China/Results" target="_blank" style="text-decoration: underline;">here<img style="height: 20px;" src="https://static.igem.org/mediawiki/2019/b/bd/T--ECUST_China--icon_cursor.png"></a>.</p>
 +
        <p>4nd, July
 +
—We successfully got all the fragments required to construct inverter dual plasmid system.  </p>
 +
        <p>10th, July
 +
—We constructed the inverter system and verified its sequence by first generation sequencing..
 +
        <p>25th,July
 +
—We got the preliminary results that the plasmid pIN2 worked as expected since the fluorescence of mRFP was detectable even with eyes.
 
         </li>
 
         </li>
 
         </ul>
 
         </ul>
 
         <h2>—August 2019</h2>
 
         <h2>—August 2019</h2>
 +
 +
        <p>28th, August
 +
—We visited to Fulen, Hangzhou, a paper recycling company to investigate the value and application of our project.
 
         <ul class="circle">
 
         <ul class="circle">
 
         <li><p><b>Cellulose degrading ability</b></p>
 
         <li><p><b>Cellulose degrading ability</b></p>
−
              <p>that</p>
+
        <p>1st, August
 +
—We successfully constructed cex / cenA / eCFP with hemolysin system on pET-28b backbone,  but saw not expression of either protein.
 +
        <p>9th, August
 +
—We ran a SDS-PAGE to detect protein expression, the eCFP and cex/cenA were all not expressed.
 +
        <p>12th, August
 +
—We suspected the failure of expression was because we changed the RBS of original pET-28b backbone during primer design. This time, we didn’t change any sequence of the backbone and chose a dual promoter to control the expression of cex/cenA and hlyABD.
 +
        <p>21th, August
 +
—We met some problems constructing this dual promoter plasmid, since there were always some trouble existing in the linkage section.
 
         </li>
 
         </li>
 
         <li><p><b>Cellulose producing ability</b></p>
 
         <li><p><b>Cellulose producing ability</b></p>

Revision as of 01:23, 22 October 2019

Introduction

Paper Transformer is a superhero we made to change the paper world, and several of team members devoted most of their time and energy this season.

We assigned four iGEMers to achieve three abilities of our Paper Transformer. In detail, we assigned Hu Shijia for cellulose degrading module, Xiang Lan for cellulose producing module, Miao Zongjie and Jin Qianwei for inverter module.

Progress

—January 2019

The senior students publicized the iGEM and recruited candidates aspiring to participate iGEM representing ECUST-2019 iGEM team.

—February 2019

We had a brain storm within the iGEM club of ECUST.

—April 2019

We participated a meetup held at New York University Shanghai.

—May 2019

We decided to focus on Paper Transformer as our project out of three others.

We were invited to Jiangnan University in Wuxi, Jiangsu province to discuss our project. We communicated with three teams of Jiangnan University on human practices and gene circuit design.

—June 2019

We started to plan our wet lab arrangement and construction work.

—July 2019

28th, July —We went to ShenZhen to participate CCiC 2019.

Our experiment didn’t start until this month, but we quickly assigned the tasks into several team member. In detail, shows as follows:

  • Cellulose degrading ability

    This part was mostly undertaken by Hushijia, and later assisted by Xianglan. The results could be seen at here.

    2nd, July —We firstly attempted to construct the cex and cenA on the pET-28b separately, since the dual inverter plasmid has not been constructed and the pET-28b is a reliable plasmid to express heterologous protein.

    9th, July —At the beginning, we got desired parts from kit by PCR very easily. And through Gibson-assembly method, we ligated the parts we purified.

    20th,July —When we tested the ligation section by test primer and first generation sequencing, nonetheless, there were not a single clone comprise all the fragments we expected to be ligated.

    Protocols

  • Cellulose producing ability

    This part was firstly undertaken by Xianglan, and then taken over by Jinqianwei and Miaozongjie. The results could be seen at here.

    1nd, July —We tried to purchase Gluconacetobacter xylinus from China General Microbiological Culture Collection Center, but we decided not to finish this order since we didn’t have the protocols and available plasmid in our school, making it almost impossible for us to set it as our chasis.

    7th, July —We chose E.coli DH5alpha as our chasis.

    18th,July —We met some problems about acquiring the acsAB and acsCD from the kit, since they were both about 5500bp.

    26th,July —Even though we occasionally got the desired fragments, problems still exist in the ligation procedure. We couldn’t ligate the acsAB, and acsCD with neither pET-28b backbone nor pSB1C3 by Gibson-assembly and T4-ligation respectively.

  • Inversion-control ability

    This part was mostly undertaken by Jinqianwei and Miaozongjie. The results could be seen at here.

    4nd, July —We successfully got all the fragments required to construct inverter dual plasmid system.

    10th, July —We constructed the inverter system and verified its sequence by first generation sequencing..

    25th,July —We got the preliminary results that the plasmid pIN2 worked as expected since the fluorescence of mRFP was detectable even with eyes.

—August 2019

28th, August —We visited to Fulen, Hangzhou, a paper recycling company to investigate the value and application of our project.

  • Cellulose degrading ability

    1st, August —We successfully constructed cex / cenA / eCFP with hemolysin system on pET-28b backbone, but saw not expression of either protein.

    9th, August —We ran a SDS-PAGE to detect protein expression, the eCFP and cex/cenA were all not expressed.

    12th, August —We suspected the failure of expression was because we changed the RBS of original pET-28b backbone during primer design. This time, we didn’t change any sequence of the backbone and chose a dual promoter to control the expression of cex/cenA and hlyABD.

    21th, August —We met some problems constructing this dual promoter plasmid, since there were always some trouble existing in the linkage section.

  • Cellulose producing ability

    that

  • Inversion-control ability

    that

—September 2019

  • Cellulose degrading ability

    that

  • Cellulose producing ability

    that

  • Inversion-control ability

    that

—October 2019

  • Cellulose degrading ability

    that

  • Cellulose producing ability

    that

  • Inversion-control ability

    that

More details about our notebook please click

Notebook




ECUST_China

EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY

Shanghai, China

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ecust_igem_2019@163.com