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

Line 203: Line 203:
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.1th</b></h1>
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.1th</b></h1>
 
<div style="padding-left:32px;"><li>A colony PCR was performed with five colonies.</li>
 
<div style="padding-left:32px;"><li>A colony PCR was performed with five colonies.</li>
2.Gel electrophoresis showed that 5 colonies were positive for insertion of <i>19</i>.<br/>
+
<li>Gel electrophoresis showed that 5 colonies were positive for insertion of <i>19</i>.</li>
3.Two of these colonies containing <i>19</i> were used to inoculate overnight cultures.<br/>
+
<li>Two of these colonies containing <i>19</i> were used to inoculate overnight cultures.</li>
4.<i>15</i> gene fragment was phosphorylated.<br/>
+
<li><i>15</i> gene fragment was phosphorylated.</li>
 
</div>
 
</div>
 
<br/>
 
<br/>
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<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.2th</b></h1>
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.2th</b></h1>
<div style="padding-left:32px;">1.Plasmids containing T vector with <i>19</i>(pT-19)were isolated using a miniprep kit.<br/>
+
<div style="padding-left:32px;"><li>Plasmids containing T vector with <i>19</i>(pT-19)were isolated using a miniprep kit.</li>
2.Mono-restriction digest of pT-19 with stu I. <br/>
+
<li>Mono-restriction digest of pT-19 with stu I. </li>
3.The enzyme-digested product was dephosphorylation.<br/>
+
<li>The enzyme-digested product was dephosphorylation.</li>
  
<img align="center" src="https://static.igem.org/mediawiki/2016/a/aa/Igem-6803-week3.png"  alt="Igem-6803-week3-2" width="60%"><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/>
 
</a>
 
</a>
  
  
4.Dephosphorylated plasmid and phosphorylated gene <i>15</i> were connected.<br/>
+
<li>Dephosphorylated plasmid and phosphorylated gene <i>15</i> were connected.</li>
5.Ligation product was transformed into E.coli via heat shock.<br/>
+
<li>Ligation product was transformed into E.coli via heat shock.</li>
 
</div>
 
</div>
 
<br/>
 
<br/>
  
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.3th</b></h1>
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.3th</b></h1>
<div style="padding-left:32px;">1.A colony PCR was performed with twelve colonies.<br/>
+
<div style="padding-left:32px;"><li>A colony PCR was performed with twelve colonies.</li>
2.Gel electrophoresis showed that 2 colonies were positive for connecting the plasmid and gene fragment.<br/>
+
<li>Gel electrophoresis showed that 2 colonies were positive for connecting the plasmid and gene fragment.</li>
3.These two colonies were used to inoculate overnight cultures.<br/>
+
<li>These two colonies were used to inoculate overnight cultures.</li>
 
</div>
 
</div>
 
<br/>
 
<br/>
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<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.4th</b></h1>
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.4th</b></h1>
<div style="padding-left:32px;">1.Plasmids with correct sequence of  <i>19-15</i>  were isolated using a miniprep kit.<br/>
+
<div style="padding-left:32px;"><li>Plasmids with correct sequence of  <i>19-15</i>  were isolated using a miniprep kit.<li>
2.Mono-restriction digest of pT-19-15 with Nru I.<br/>
+
<li>Mono-restriction digest of pT-19-15 with Nru I.</li>
3.The enzyme-digested product was dephosphorylation.<br/>
+
<li>The enzyme-digested product was dephosphorylation.</li>
 
</div>
 
</div>
 
<br/>
 
<br/>
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<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.7th</b></h1>
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.7th</b></h1>
<div style="padding-left:32px;">1.Plasmids were isolated using a miniprep kit.<br/>
+
<div style="padding-left:32px;"><li>Plasmids were isolated using a miniprep kit.</li>
2.Amplification of <i>13</i> with Q5 High-Fidelity DNA Polymerase out of E.coli <br/>
+
<li>Amplification of <i>13</i> with Q5 High-Fidelity DNA Polymerase out of E.coli </li>
3.<i>13</i> amplification at 65.0°C with 13.rev/fwd primes<br/>
+
<li><i>13</i> amplification at 65.0°C with 13.rev/fwd primes</li>
 
&nbsp;&nbsp;PCR worked, positive control worked, no amplification of <i>13</i><br/>
 
&nbsp;&nbsp;PCR worked, positive control worked, no amplification of <i>13</i><br/>
4.The fragments of <i>13</i> were purified with PCR Purification Kit.<br/>
+
<li>The fragments of <i>13</i> were purified with PCR Purification Kit.</li>
 
</div>
 
</div>
 
<br/>
 
<br/>
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<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.9th</b></h1>
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.9th</b></h1>
<div style="padding-left:32px;">1.Insertion of Ni promoter and ligation of <i>13-19-15</i><br/>
+
<div style="padding-left:32px;"><li>Insertion of Ni promoter and ligation of <i>13-19-15</i></li>
2.Ni inducible promoter was ligated into pCPC-3301 vector.<br/>
+
<li>Ni inducible promoter was ligated into pCPC-3301 vector.</li>
3.Phosphorylated <i>13</i> was ligated into pT-19-15 and transformed into E.coli via heat shock.<br/>
+
<li>Phosphorylated <i>13</i> was ligated into pT-19-15 and transformed into E.coli via heat shock.</li>
4.Single colonies were obtained by plating.<br/>
+
<li>Single colonies were obtained by plating.</li>
 
</div>
 
</div>
 
<br/>
 
<br/>
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<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.10th</b></h1>
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.10th</b></h1>
 
<div style="padding-left:32px;">
 
<div style="padding-left:32px;">
1.A colony PCR of Ni inducible promoter(pCPC-3301-Ni) was performed with 12 colonies.<br/>
+
<li>A colony PCR of Ni inducible promoter(pCPC-3301-Ni) was performed with 12 colonies.<li>
 
&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/>
2.A colony PCR of pT-13-19-15 was performed with 7 colonies.<br/>
+
<li>A colony PCR of pT-13-19-15 was performed with 7 colonies.<li>
 
&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/>
  
<img align="center" src="https://static.igem.org/mediawiki/2016/8/81/Igem-6803-week4.png"  alt="Igem-6803-week4" width="500px"> <br/>
+
<img align="center" src="https://static.igem.org/mediawiki/2016/8/81/Igem-6803-week4.png"  alt="Igem-6803-week4" width="800" height="533"> <br/>
 
</a>
 
</a>
  
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<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.15th</b></h1>
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.15th</b></h1>
<div style="padding-left:32px;">1.Colonies containing Ni inducible promoter were used to inoculate overnight cultures.<br/>
+
<div style="padding-left:32px;"><li>Colonies containing Ni inducible promoter were used to inoculate overnight cultures.</li>
2.PCR was performed to check if the gene fragments were ligated correctly.<br/>
+
<li>PCR was performed to check if the gene fragments were ligated correctly.</li>
 
&nbsp;&nbsp;13_ fwd and 15_rev on pT-13-19-15<br/>
 
&nbsp;&nbsp;13_ fwd and 15_rev on pT-13-19-15<br/>
3.Gel electrophoresis showed that it failed.<br/>
+
<li>Gel electrophoresis showed that it failed.</li>
 
</div>
 
</div>
 
<br/>
 
<br/>
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<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.16th</b></h1>
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.16th</b></h1>
<div style="padding-left:32px;">Plasmids pCPC-3031-Ni were isolated using a miniprep kit.<br/>
+
<div style="padding-left:32px;"><li>Plasmids pCPC-3031-Ni were isolated using a miniprep kit.</li>
 
</div>
 
</div>
 
<br/>
 
<br/>
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<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.17th</b></h1>
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.17th</b></h1>
 
<div style="padding-left:32px;">
 
<div style="padding-left:32px;">
1.Several PCRs were performed to check if the gene fragments were ligated correctly.<br/>
+
<li>Several PCRs were performed to check if the gene fragments were ligated correctly.</li>
 
&nbsp;&nbsp;13_fwd and 13_rev on pT-13-19-15<br/>
 
&nbsp;&nbsp;13_fwd and 13_rev on pT-13-19-15<br/>
 
&nbsp;&nbsp;19_fwd and 19_rev on pT-13-19-15<br/>
 
&nbsp;&nbsp;19_fwd and 19_rev on pT-13-19-15<br/>
Line 383: Line 383:
 
</a>
 
</a>
  
2.<b>Repetition:</b>  Phosphorylated <i>13</i> was ligated into pT-19-15 and transformed into E.coli via heat shock.<br/>
+
<li><b>Repetition:</b>  Phosphorylated <i>13</i> was ligated into pT-19-15 and transformed into E.coli via heat shock.</li>
 
</div>
 
</div>
 
<br/>
 
<br/>
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<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.21th</b></h1>
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.21th</b></h1>
<div style="padding-left:32px;">1.Medium preparation :BG-11.<br/>
+
<div style="padding-left:32px;"><li>Medium preparation :BG-11.</li>
2.19_ fwd and 15_rev were used to amplify <i>19-15</i>.
+
<li>19_ fwd and 15_rev were used to amplify <i>19-15</i>.</li>
<br/>
+
 
 
</div>
 
</div>
 
<br/>
 
<br/>
  
 
<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;">1.Gel electrophoresis showed that amplification of fragments was successfull.<br/>
+
<div style="padding-left:32px;"><li>Gel electrophoresis showed that amplification of fragments was successfull.</li>
2.Ligated <i>13</i> and <i>19-15</i> via overlap PCR.<br/>
+
<li>Ligated <i>13</i> and <i>19-15</i> via overlap PCR.</li>
3.This PCR worked well and the products were extracted from the gel using a Agarose Gel DNA Extration Kit.<br/>
+
<li>This PCR worked well and the products were extracted from the gel using a Agarose Gel DNA Extration Kit.</li>
4.Restriction digest on pCPC-3031-Ni with Sac I.<br/>
+
<li>Restriction digest on pCPC-3031-Ni with Sac I.</li>
  
 
<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/>
 
<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/>
  
5.The fragment <i>13-19-15</i> was ligated onto T vector and transformed into E.coli via heat shock.<br/>
+
<li>The fragment <i>13-19-15</i> was ligated onto T vector and transformed into E.coli via heat shock.</li>
  
 
<img align="center" src="https://static.igem.org/mediawiki/2016/0/06/Igem-6803-week6.jpg"  alt="Igem-6803-week6-2" width="300px" ><br/>
 
<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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<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.23th</b></h1>
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.23th</b></h1>
<div style="padding-left:32px;">1.A colony PCR of pT-13-19-15 was performed with 12 colonies.<br/>
+
<div style="padding-left:32px;"><li>A colony PCR of pT-13-19-15 was performed with 12 colonies.</li>
2.Two of these colonies containing pT-13-19-15 were used to inoculate overnight cultures.<br/>
+
<li>Two of these colonies containing pT-13-19-15 were used to inoculate overnight cultures.</li>
3.<i>13-19-15</i> gene fragment was phosphorylated.<br/>
+
<li><i>13-19-15</i> gene fragment was phosphorylated.</li>
4.Phosphorylated <i>13-19-15</i> was ligated into pCPC-3031-Ni and transformed into E.coli via heat shock.<br/>
+
<li>Phosphorylated <i>13-19-15</i> was ligated into pCPC-3031-Ni and transformed into E.coli via heat shock.</li>
 
</div>
 
</div>
 
<br/>
 
<br/>
Line 459: Line 459:
  
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.24th</b></h1>
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.24th</b></h1>
<div style="padding-left:32px;">Plasmids pT-13-19-15 were isolated using a miniprep kit.<br/>
+
<div style="padding-left:32px;"><li>Plasmids pT-13-19-15 were isolated using a miniprep kit.</li>
 
</div>
 
</div>
 
<br/>
 
<br/>
Line 493: Line 493:
  
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.25th</b></h1>
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.25th</b></h1>
<div style="padding-left:32px;">1.A colony PCR of pCPC-3031-Ni-13-19-15 was performed with 12 colonies.<br/>
+
<div style="padding-left:32px;"><li>A colony PCR of pCPC-3031-Ni-13-19-15 was performed with 12 colonies.</li>
2.Three colonies were proved to be true. And two of them were used to inoculate overnight cultures.<br/>
+
<li>Three colonies were proved to be true. And two of them were used to inoculate overnight cultures.</li>
  
 
<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/>
Line 503: Line 503:
  
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.26th</b></h1>
 
<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.26th</b></h1>
<div style="padding-left:32px;">Plasmids pT-13-19-15 were handed in for sequencing, which confirmed its correctness.<br/>
+
<div style="padding-left:32px;"><li>Plasmids pT-13-19-15 were handed in for sequencing, which confirmed its correctness.</li>
 
<b>The sequencing results for them were correct.</b>
 
<b>The sequencing results for them were correct.</b>
 
<br/>
 
<br/>

Revision as of 06:52, 14 October 2016

TEAM TIANJIN


Team Tianjin-Attribution

Notes For Modified Cyanobacteria:A Controllable Lipid Producer

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.12th

    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.

    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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