Difference between revisions of "Team:Tianjin/Note/6803"

 
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<!------------------------------------------- This part should be dismissed in WIKI u
 
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<article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
 
<article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
 
<header class="entry-header">
 
<header class="entry-header">
<div class="entry-title" align="center" >Notes For Modified Cyanobacteria:A Controllable Lipid Producer</div>
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<div class="entry-title" align="center" >Notes For Cyanobacteria</div>
 
</header><!-- .entry-header -->
 
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<b><h1 style="font-size:108%">&nbsp;&nbsp;Jul.31th</b></h1>
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Jul.31th</b></h1>
 
<div style="padding-left:32px;"><li>Cultivation: 30µL mother liquid of Synechococcus sp PCC 7942 (wild type)were cultivated in 10 mL BG-11-media at 30°C and 140rpm.</li>
 
<div style="padding-left:32px;"><li>Cultivation: 30µL mother liquid of Synechococcus sp PCC 7942 (wild type)were cultivated in 10 mL BG-11-media at 30°C and 140rpm.</li>
 +
 +
 
<li>The genome DNA of 7942 was extracted using a Genomic DNA Isolation Kit.</li></div><br/>
 
<li>The genome DNA of 7942 was extracted using a Genomic DNA Isolation Kit.</li></div><br/>
  
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<div style="padding-left:32px;"><li>Synechococcus sp PCC 7942 had no signals of life.</li></div><br/>  
 
<div style="padding-left:32px;"><li>Synechococcus sp PCC 7942 had no signals of life.</li></div><br/>  
  
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<a href="https://static.igem.org/mediawiki/2016/3/37/Igem--6803-week2.jpg" data-lightbox="no" data-title="7942">
 
<img align="center" src="https://static.igem.org/mediawiki/2016/3/37/Igem--6803-week2.jpg"  alt="Igem-6803-week2" width="300px"><br/><br/>
 
<img align="center" src="https://static.igem.org/mediawiki/2016/3/37/Igem--6803-week2.jpg"  alt="Igem-6803-week2" width="300px"><br/><br/>
 
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<div style="padding-left:32px;"><li>pMV-G19 and pMV-G15  containing our target genes were received.</li><br/>
 
<div style="padding-left:32px;"><li>pMV-G19 and pMV-G15  containing our target genes were received.</li><br/>
  
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<a href="https://static.igem.org/mediawiki/2016/6/6d/Igem-6803-week3-1.jpg" data-lightbox="no" data-title="pMV-G19 and pMV-G15">
 
<img align="center" src="https://static.igem.org/mediawiki/2016/6/6d/Igem-6803-week3-1.jpg"  alt="Igem-6803-week3-1" width="300px" ><br/><br/>
 
<img align="center" src="https://static.igem.org/mediawiki/2016/6/6d/Igem-6803-week3-1.jpg"  alt="Igem-6803-week3-1" width="300px" ><br/><br/>
 
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<li>The enzyme-digested product was dephosphorylation.</li>
 
<li>The enzyme-digested product was dephosphorylation.</li>
 
<br/>
 
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<img align="center" src="https://static.igem.org/mediawiki/2016/a/aa/Igem-6803-week3.png"  alt="Igem-6803-week3-2" width="800" height="533"><br/>
 
<img align="center" src="https://static.igem.org/mediawiki/2016/a/aa/Igem-6803-week3.png"  alt="Igem-6803-week3-2" width="800" height="533"><br/>
 
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&nbsp;&nbsp;Two of the successful ones were used to inoculate overnight cultures.<br/>
 
&nbsp;&nbsp;Two of the successful ones were used to inoculate overnight cultures.<br/>
 
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<b>The fourth and sixth ones were not successful.</b><br/>
 
<b>The fourth and sixth ones were not successful.</b><br/>
 
<br/>
 
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<img align="center"src="https://static.igem.org/mediawiki/2016/4/40/Igem-6803-week5.png"  alt="Igem-6803-week5" width="800" height="533" ><br/>
 
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         <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
 
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<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.22th</b></h1>
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.22th</b></h1>
<div style="padding-left:32px;"><li>Gel electrophoresis showed that amplification of fragments was successfull.</li>
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<div style="padding-left:32px;">
 +
<li>Gel electrophoresis showed that amplification of fragments was successfull.</li>
 
<li>Ligated <i>13</i> and <i>19-15</i> via overlap PCR.</li>
 
<li>Ligated <i>13</i> and <i>19-15</i> via overlap PCR.</li>
 
<li>This PCR worked well and the products were extracted from the gel using a Agarose Gel DNA Extration Kit.</li>
 
<li>This PCR worked well and the products were extracted from the gel using a Agarose Gel DNA Extration Kit.</li>
 
<li>Restriction digest on pCPC-3031-Ni with Sac I.</li>
 
<li>Restriction digest on pCPC-3031-Ni with Sac I.</li>
 
<br/>
 
<br/>
<img align="center" src="https://static.igem.org/mediawiki/2016/8/89/Igem-6803-week6-2.jpg"  alt="Igem-6803-week6-1" width="300px"><br/>
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<a href="https://static.igem.org/mediawiki/2016/8/89/Igem-6803-week6-2.jpg" data-lightbox="no" data-title=" ">
<br/>
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<img align="center" src="https://static.igem.org/mediawiki/2016/8/89/Igem-6803-week6-2.jpg"  alt="Igem-6803-week6-1" width="300px">
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</a>
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<li>The fragment <i>13-19-15</i> was ligated onto T vector and transformed into E.coli via heat shock.</li>
 
<li>The fragment <i>13-19-15</i> was ligated onto T vector and transformed into E.coli via heat shock.</li>
<br/>
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<img align="center" src="https://static.igem.org/mediawiki/2016/0/06/Igem-6803-week6.jpg"  alt="Igem-6803-week6-2" width="300px" ><br/>
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<img align="center" src="https://static.igem.org/mediawiki/2016/0/06/Igem-6803-week6.jpg"  alt="Igem-6803-week6-2" width="300px" >
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         <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
 
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<li>Three colonies were proved to be true. And two of them were used to inoculate overnight cultures.</li>
 
<li>Three colonies were proved to be true. And two of them were used to inoculate overnight cultures.</li>
 
<br/>
 
<br/>
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<a href="https://static.igem.org/mediawiki/2016/2/2d/Igem-6803-week6-3.png" data-lightbox="no" data-title=" ">
 
<img align="center" src="https://static.igem.org/mediawiki/2016/2/2d/Igem-6803-week6-3.png"  alt="Igem-6803-week6-3" width="800" height="533" ><br/>
 
<img align="center" src="https://static.igem.org/mediawiki/2016/2/2d/Igem-6803-week6-3.png"  alt="Igem-6803-week6-3" width="800" height="533" ><br/>
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<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.26th</b></h1>
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.26th</b></h1>
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<br/>
 
<br/>
 
</div>
 
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<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.28th</b></h1>
 +
<div style="padding-left:32px;"><li>We transformed the expression vector, pCPC-3031-Ni-13-19-15, into <i>Synechocystis 6803 </i>via electroporation.</li>
 
<br/>
 
<br/>
 +
<b>The story of 6803 is not over. Other team members will show the completed process during the presentation. </br>
 +
I will cherish the time spending with you for my whole life. ———by Jiawei Chen and Liangyu Qian</b>
 +
</div>
  
  
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</div>
 
</div>
<div class="side-nav">
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<ul>
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<li><a class="topLink" href="#Topp" style="color:#5555FF">Top</a></li>
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                                                        <li><a class="topLink" href="#Week1" style="color:#5555FF">Week1</a></li>
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<li><a class="topLink" href="#Week2" style="color:#5555FF">Week2</a></li>
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<li><a class="topLink" href="#Week3" style="color:#5555FF">Week3</a></li>
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<li><a class="topLink" href="#Week4" style="color:#5555FF">Week4</a></li>
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<li><a class="topLink" href="#Week5" style="color:#5555FF">Week5</a></li>
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<li><a class="topLink" href="#Week6" style="color:#5555FF">Week6</a></li>
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<li><a class="topLink" href="#Week7" style="color:#5555FF">Week7</a></li>
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Latest revision as of 10:44, 5 November 2016

TEAM TIANJIN


Team Tianjin-Attribution

Notes For Cyanobacteria

Week1(7/31/2016-8/6/2016)

  Jul.31th

  • Cultivation: 30µL mother liquid of Synechococcus sp PCC 7942 (wild type)were cultivated in 10 mL BG-11-media at 30°C and 140rpm.
  • The genome DNA of 7942 was extracted using a Genomic DNA Isolation Kit.

  • Show More

    Week2(8/14/2016-8/20/2016)

  • Synechococcus sp PCC 7942 had no signals of life.

  • Igem-6803-week2


    Show More

    Week3(8/28/2016-9/3/2016)

      Aug.29th

  • pMV-G19 and pMV-G15 containing our target genes were received.

  • Igem-6803-week3-1


  • Colonies were used to inoculate overnight cultures.

  •   Aug.30th

  • Plasmids were isolated using a miniprep kit.
  • Amplification of 19 and 15 with Q5 High-Fidelity DNA Polymerase out of E.coli.
  •   19 amplification at 65.0°C with 19.rev/fwd primes.
            PCR worked, positive control worked, no amplification of 19.
      15 amplification at 65.0°C with 15.rev/fwd primes.
            PCR worked, positive control worked, no amplification of 15.
  • A product length of 900 bp was expected. The gel shows that most plasmids seem to be correct.
  •   This result was confirmed by sequencing.
  • The fragments of 19 and 15 were purified with PCR Purification Kit.

  •   Aug.31th

  • Ligation of 15 with 19.
  • We add a single 3`-adenine overhang to each end of the fragment of 19 and then purified with DNA Purification Kit.
  • 19 was ligated into T vectors via TA clone and transformed into E.coli via heat shock.

  •   Sep.1th

  • A colony PCR was performed with five colonies.
  • Gel electrophoresis showed that 5 colonies were positive for insertion of 19.
  • Two of these colonies containing 19 were used to inoculate overnight cultures.
  • 15 gene fragment was phosphorylated.

  •   Sep.2th

  • Plasmids containing T vector with 19(pT-19)were isolated using a miniprep kit.
  • Mono-restriction digest of pT-19 with stu I.
  • The enzyme-digested product was dephosphorylation.

  • Igem-6803-week3-2

  • Dephosphorylated plasmid and phosphorylated gene 15 were connected.
  • Ligation product was transformed into E.coli via heat shock.

  •   Sep.3th

  • A colony PCR was performed with twelve colonies.
  • Gel electrophoresis showed that 2 colonies were positive for connecting the plasmid and gene fragment.
  • These two colonies were used to inoculate overnight cultures.

  • Show More

    Week4(9/4/2016-9/10/2016)

      Sep.4th

  • Plasmids with correct sequence of 19-15 were isolated using a miniprep kit.
  • Mono-restriction digest of pT-19-15 with Nru I.
  • The enzyme-digested product was dephosphorylation.

  •   Sep.6th

  • Colonies containing gene 13 were used to inoculate overnight cultures.

  •   Sep.7th

  • Plasmids were isolated using a miniprep kit.
  • Amplification of 13 with Q5 High-Fidelity DNA Polymerase out of E.coli
  • 13 amplification at 65.0°C with 13.rev/fwd primes
  •   PCR worked, positive control worked, no amplification of 13
  • The fragments of 13 were purified with PCR Purification Kit.

  •   Sep.8th

  • 13 gene fragment was phosphorylated.

  •   Sep.9th

  • Insertion of Ni promoter and ligation of 13-19-15
  • Ni inducible promoter was ligated into pCPC-3301 vector.
  • Phosphorylated 13 was ligated into pT-19-15 and transformed into E.coli via heat shock.
  • Single colonies were obtained by plating.

  •   Sep.10th

  • A colony PCR of Ni inducible promoter(pCPC-3301-Ni) was performed with 12 colonies.
  •   Two of the successful ones were used to inoculate overnight cultures.
  • A colony PCR of pT-13-19-15 was performed with 7 colonies.
  •   Two of the successful ones were used to inoculate overnight cultures.

    Igem-6803-week4


  • Show More

    Week5(9/11/2016-9/17/2016)

      Sep.11th

  • Two kinds of plasmids were isolated using a miniprep kit.

  •   Sep.14th

  • The sequencing results for both of them were error.

  •   Sep.15th

  • Colonies containing Ni inducible promoter were used to inoculate overnight cultures.
  • PCR was performed to check if the gene fragments were ligated correctly.
  •   13_ fwd and 15_rev on pT-13-19-15
  • Gel electrophoresis showed that it failed.

  •   Sep.16th

  • Plasmids pCPC-3031-Ni were isolated using a miniprep kit.

  •   Sep.17th

  • Several PCRs were performed to check if the gene fragments were ligated correctly.
  •   13_fwd and 13_rev on pT-13-19-15
      19_fwd and 19_rev on pT-13-19-15
      15_fwd and 15_rev on pT-13-19-15
      13_fwd and 19_rev on pT-13-19-15
      19_fwd and 15_rev on pT-13-19-15
      13_ wd and 15_rev on pT-13-19-15
    The fourth and sixth ones were not successful.

    Igem-6803-week5

  • Repetition: Phosphorylated 13 was ligated into pT-19-15 and transformed into E.coli via heat shock.

  • Show More

    Week6(9/18/2016-9/24/2016)

      Sep.18th

    Repetition: Several PCRs were performed to check if the gene fragments were ligated correctly.
      13_fwd and 13_rev on pT-13-19-15
      13_fwd and 19_rev on pT-13-19-15
    The second one was failed.

      Sep.21th

  • Medium preparation :BG-11.
  • 19_ fwd and 15_rev were used to amplify 19-15.

  •   Sep.22th

  • Gel electrophoresis showed that amplification of fragments was successfull.
  • Ligated 13 and 19-15 via overlap PCR.
  • This PCR worked well and the products were extracted from the gel using a Agarose Gel DNA Extration Kit.
  • Restriction digest on pCPC-3031-Ni with Sac I.

  • Igem-6803-week6-1
  • The fragment 13-19-15 was ligated onto T vector and transformed into E.coli via heat shock.
  • Igem-6803-week6-2

      Sep.23th

  • A colony PCR of pT-13-19-15 was performed with 12 colonies.
  • Two of these colonies containing pT-13-19-15 were used to inoculate overnight cultures.
  • 13-19-15 gene fragment was phosphorylated.
  • Phosphorylated 13-19-15 was ligated into pCPC-3031-Ni and transformed into E.coli via heat shock.

  •   Sep.24th

  • Plasmids pT-13-19-15 were isolated using a miniprep kit.

  • Show More

    Week7(9/25/2016-10/1/2016)

      Sep.25th

  • A colony PCR of pCPC-3031-Ni-13-19-15 was performed with 12 colonies.
  • Three colonies were proved to be true. And two of them were used to inoculate overnight cultures.

  • Igem-6803-week6-3


      Sep.26th

  • Plasmids pT-13-19-15 were handed in for sequencing, which confirmed its correctness.
  • The sequencing results for them were correct.

      Sep.28th

  • We transformed the expression vector, pCPC-3031-Ni-13-19-15, into Synechocystis 6803 via electroporation.

  • The story of 6803 is not over. Other team members will show the completed process during the presentation.
    I will cherish the time spending with you for my whole life. ———by Jiawei Chen and Liangyu Qian
    Show More
    info_outline
    Notice: This page is currently under construction. Contents in this page are temporaory and will be modified several times before the final release.     — 2016 iGEM Team Tianjin


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