Difference between revisions of "Team:York/Notebook"

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<p>The following is a weekly description of the experiments carried out each week. To see the protocols click <a href="https://2015.igem.org/Team:York/Protocols">here</a></p>
 
<p>The following is a weekly description of the experiments carried out each week. To see the protocols click <a href="https://2015.igem.org/Team:York/Protocols">here</a></p>
  
−
<h3>Dry Lab Work Period</h3>
+
<div class="col-md-3">
−
<ul>
+
<div class="navbar">
−
<li>28/01: Brief introductory meeting about the iGEM competition
+
    <ul>
−
<li>17/03: The official iGEM team to represent the University of York was formed!
+
        <li style="display:block;" onclick="toggleWeek('DryLab')">Dry Lab Work Period</li>
−
<li>Daily dry lab research begins, primer and construct designs made and ordered.</li>
+
        <li style="display:block;" onclick="toggleWeek('Week1')">Week 1</li>
−
<li>05/05: <a href="http://www.nouse.co.uk/2015/05/05/money-saving-micro-organisms/">Article</a>published in Nouse (University newspaper) about the 2015 iGEM team.
+
        <li style="display:block;" onclick="toggleWeek('Week2')">Week 2</li>
−
<li>03/06: We were invited to Science and Technology Alumni Network discussion event (outreach)
+
        <li style="display:block;" onclick="toggleWeek('Week3')">Week 3</li>
−
<li>04/06: <a href="https://www.york.ac.uk/biology/news-events/other/igem2015/">Article</a>published on the University's central news about this years iGEM team.
+
        <li style="display:block;" onclick="toggleWeek('Week4')">Week 4</li>
−
<li>12/06: A meeting was held with Yorkshire Water to discuss water remediation processes in the Yorkshire region
+
        <li style="display:block;" onclick="toggleWeek('Week5')">Week 5</li>
−
<li>13/06: We were invited to a University of York Biology Alumni event
+
        <li style="display:block;" onclick="toggleWeek('Week6')">Week 6</li>
−
<li>15/06:
+
        <li style="display:block;" onclick="toggleWeek('Week7')">Week 7</li>
−
<ol>
+
        <li style="display:block;" onclick="toggleWeek('Week8')">Week 8</li>
−
<li>Some of us went to a round-table discussion about the impact of Santander at York as some of the team members won the International Connections Award by Santander.</li>
+
        <li style="display:block;" onclick="toggleWeek('Week9')">Week 9</li>
−
<li><a href="http://mindthehorizon.com/2015/06/15/igem-genetic-engineering-future/">Article</a> published on Mind the Horizon about University of York iGEM</li></ol></li>
+
        <li style="display:block;" onclick="toggleWeek('Week10')">Week 10</li>
−
<li>19/06:
+
        <li style="display:block;" onclick="toggleWeek('Week11')">Week 11</li>
−
  <ol>
+
        <li style="display:block;" onclick="toggleWeek('Week12')">Week 12</li>
−
<li> Lab safety induction was carried out</li>
+
        <li style="display:block;" onclick="toggleWeek('Week13')">Week 13</li>
−
<li>We passed out pamphlets at the York Festival of Ideas to raise awareness about the iGEM competition and synthetic biology.</li></ol></li>
+
    </ul>
−
<li> 26&27/06: We participated in giving talks to prospective students about iGEM.</li>
+
</div>
−
</ul>  
+
</div>
  
−
<h3>Week 1 - June 23-27 </h3>
+
<div class="col-md-9">
−
<ul>
+
<div class="DatePeriod" id="DryLab">
−
<li>24/07: Wet lab practice day- reviewed basic lab protocols- making LB, agar plates, competent cells, transformations, PCR </li>
+
  <h3>Dry Lab Work Period</h3>
−
</ul>
+
  <ul>
 +
  <li>28/01: Brief introductory meeting about the iGEM competition
 +
  <li>17/03: The official iGEM team to represent the University of York was formed!
 +
  <li>Daily dry lab research begins, primer and construct designs made and ordered.</li>
 +
  <li>05/05: <a href="http://www.nouse.co.uk/2015/05/05/money-saving-micro-organisms/">Article</a>published in Nouse (University newspaper) about the 2015 iGEM team.
 +
  <li>03/06: We were invited to Science and Technology Alumni Network discussion event (outreach)
 +
  <li>04/06: <a href="https://www.york.ac.uk/biology/news-events/other/igem2015/">Article</a>published on the University's central news about this years iGEM team.
 +
  <li>12/06: A meeting was held with Yorkshire Water to discuss water remediation processes in the Yorkshire region
 +
  <li>13/06: We were invited to a University of York Biology Alumni event
 +
  <li>15/06:
 +
  <ol>
 +
  <li>Some of us went to a round-table discussion about the impact of Santander at York as some of the team members won the International Connections Award by Santander.</li>
 +
  <li><a href="http://mindthehorizon.com/2015/06/15/igem-genetic-engineering-future/">Article</a> published on Mind the Horizon about University of York iGEM</li></ol></li>
 +
  <li>19/06:
 +
    <ol>
 +
  <li> Lab safety induction was carried out</li>
 +
  <li>We passed out pamphlets at the York Festival of Ideas to raise awareness about the iGEM competition and synthetic biology.</li></ol></li>
 +
  <li> 26&27/06: We participated in giving talks to prospective students about iGEM.</li>
 +
  </ul>
 +
</div>
  
−
<h3>Week 2 - June 29- July 3 </h3>
+
<div class="DatePeriod" id="Week1">
−
<ul>
+
  <h3>Week 1 - June 23-27 </h3>
−
<li>01/07:Competent cells made</li>
+
  <ul>
−
<li>02/07:Plates were streaked with two isolates of each deletion wanted from the Keio collection [PPX, PPK, PstA, PstB, PstC, PstS and PhoE] These have been collected and put in the incubating (37°C) room.</li>
+
  <li>24/07: Wet lab practice day- reviewed basic lab protocols- making LB, agar plates, competent cells, transformations, PCR </li>
−
<li>03/07:Competent cells were tested with iGEM transformation efficiency kit</li>
+
  </ul>
−
</ul>
+
</div>
  
−
<h3>Week 3 - July 6-10 </h3>
+
<div class="DatePeriod" id="Week2">
−
<ul>
+
  <h3>Week 2 - June 29- July 3 </h3>
−
<li>07/07: More agar plates made, next set of competent cell procedure started</li>
+
  <ul>
−
<li>07/07: YUfund page done and submitted</li>
+
  <li>01/07:Competent cells made</li>
−
</ul>
+
  <li>02/07:Plates were streaked with two isolates of each deletion wanted from the Keio collection [PPX, PPK, PstA, PstB, PstC, PstS and PhoE] These have been collected and put in the incubating (37°C) room.</li>
 +
  <li>03/07:Competent cells were tested with iGEM transformation efficiency kit</li>
 +
  </ul>
 +
</div>
  
−
<h3>Week 4 - July 13-17  </h3>
+
<div class="DatePeriod" id="Week3">
−
<ul>
+
  <h3>Week 3 - July 6-10 </h3>
−
<li>14/07: Vistited Badger Hill Primary School and held a bacteria workshop for the students</li>
+
  <ul>
−
<li>15/07: first day of demonstrations of cell transformations and aseptic technique to sixth formers</li>
+
  <li>07/07: More agar plates made, next set of competent cell procedure started</li>
−
<li>16/07: second day of demonstrations to sixth formers:analysing transformations and building bioreactors</li>
+
  <li>07/07: YUfund page done and submitted</li>
−
<Li>17/07: analysing bioreactors with sixth formers</li>
+
  </ul>
−
<li>17/07: phosphate assay done to get a standard curve</li>
+
</div>
−
<li>17/07: first attempt at growth assay - had to be redone, as computer crashed and updated mid-run</li>
+
−
</ul>
+
  
−
<h3>Week 5 - July 20-24 </h3>
+
<div class="DatePeriod" id="Week4">
−
<ul>
+
  <h3>Week 4 - July 13-17 </h3>
−
<li>20/07:Phosphate assay was started for KEIO collection - PPK, PPX, wildtype - no results</li>
+
  <ul>
−
<li>20/07:Colony PCR on SmPstSCAB, EcPstSCAB and EcPhoE</li>
+
  <li>14/07: Vistited Badger Hill Primary School and held a bacteria workshop for the students</li>
−
<li>20/07:Next set of competent cells made </li>
+
  <li>15/07: first day of demonstrations of cell transformations and aseptic technique to sixth formers</li>
−
<li>22/07:X-Gal/IPTG Plates made </li>
+
  <li>16/07: second day of demonstrations to sixth formers:analysing transformations and building bioreactors</li>
−
<li>22/07:Digested pSB1C3 with EcoRI and DpnI, then used Gibson Assembly to insert our adapter genes in.
+
  <Li>17/07: analysing bioreactors with sixth formers</li>
−
<li>23/07:Visit to AgeUK LunchClub to explain about the GM</li>
+
  <li>17/07: phosphate assay done to get a standard curve</li>
−
<li>23/07:Glycerol stocks of <em>Sinorhizobium meliloti</em></li>
+
  <li>17/07: first attempt at growth assay - had to be redone, as computer crashed and updated mid-run</li>
−
<li>23/07:Phosphate assay of PPK, PPX, wildtype again with formic acid. Samples were neutralised before plating. - no results </li>
+
  </ul>
−
<li>24/07:Ran gel electrophoresis of PCR from 22/07</li>
+
</div>
−
<li>24/07:Second attempt at growth assay - had to be redone due to poor cell growth, potentially caused by poor spreading of plates</li>
+
−
</ul>
+
  
−
<h3>Week 6 - July 27-31 </h3>
+
<div class="DatePeriod" id="Week5">
−
<ul>
+
  <h3>Week 5 - July 20-24 </h3>
−
<li>27/07:Preparations for glassmilk procedure</li>
+
  <ul>
−
<li>28/07:First day of outreaching event to Saint Helen's CHurch, York, to carry out strawberry extraction practical and briefly explain about the steps involved for GM </li>
+
  <li>20/07:Phosphate assay was started for KEIO collection - PPK, PPX, wildtype - no results</li>
−
<li>29/07:Second (and the last day) of outreaching event to Saint Helen's Church, York, to carry out strawberry extraction practical and briefly explain about the steps involved for GM </li>
+
  <li>20/07:Colony PCR on SmPstSCAB, EcPstSCAB and EcPhoE</li>
−
<li>30/07:After several repeats the phosphate assay team managed to get significant data</li>
+
  <li>20/07:Next set of competent cells made </li>
−
<li>30/07:Phosphate assay on Keasling strains: pBC9, pBC29 and pKDM12.</li>
+
  <li>22/07:X-Gal/IPTG Plates made </li>
−
</ul>
+
  <li>22/07:Digested pSB1C3 with EcoRI and DpnI, then used Gibson Assembly to insert our adapter genes in.
 +
  <li>23/07:Visit to AgeUK LunchClub to explain about the GM</li>
 +
  <li>23/07:Glycerol stocks of <em>Sinorhizobium meliloti</em></li>
 +
  <li>23/07:Phosphate assay of PPK, PPX, wildtype again with formic acid. Samples were neutralised before plating. - no results </li>
 +
  <li>24/07:Ran gel electrophoresis of PCR from 22/07</li>
 +
  <li>24/07:Second attempt at growth assay - had to be redone due to poor cell growth, potentially caused by poor spreading of plates</li>
 +
  </ul>
 +
</div>
  
−
<h3>Week 7 - August 3-7 </h3>
+
<div class="DatePeriod" id="Week6">
−
<ul>
+
  <h3>Week 6 - July 27-31 </h3>
−
<li>03/08:Plasmid (pBC 29 Keasling collection )loss experiment initiated</li>
+
  <ul>
−
<li>05/08:Q5 PCR of SmPst, EcPst, EcPhoE</li>
+
  <li>27/07:Preparations for glassmilk procedure</li>
−
<li>17/08:Third attempt at growth assay - did not test any Pst or PhoE genes, results ok</li>
+
  <li>28/07:First day of outreaching event to Saint Helen's CHurch, York, to carry out strawberry extraction practical and briefly explain about the steps involved for GM </li>
−
<li>06/08:Inoculated overnight cultures of ApPst, ApPPK (SK-12, BA-91, UW-1)</li>
+
  <li>29/07:Second (and the last day) of outreaching event to Saint Helen's Church, York, to carry out strawberry extraction practical and briefly explain about the steps involved for GM </li>
−
<li>06/08:Ran GoTaq Colony PCR of same colonies of ApPst, ApPPK (SK-12, BA-91, UW-1)</li>
+
  <li>30/07:After several repeats the phosphate assay team managed to get significant data</li>
−
<li>06/08:Ran gel electrophoresis of colony PCR</li>
+
  <li>30/07:Phosphate assay on Keasling strains: pBC9, pBC29 and pKDM12.</li>
−
<li>06/08:Inoculated overnight cultures of BW25113, pkDM12, KoPPK</li>
+
  </ul>
−
<li>17/08:Fourth attempt at growth assay - exact repeat to verify results</li>
+
</div>
−
<li>07/08:Purified plasmids from overnight cultures of ApPst, ApPPK (SK-12, BA-91, UW-1)- "mini-prep"</li>
+
−
<li>07/08:Used Nanodrop machine to measure concentration of miniprep products</li>
+
−
<li>07/08:Performed an analytical digest of purified plasmids with EcoR1 and Pst1</li>
+
−
</ul>
+
  
−
<h3>Week 8 - August 10-14    </h3>
+
<div class="DatePeriod" id="Week7">
−
<ul>
+
  <h3>Week 7 - August 3-7  </h3>
−
<li>10/08:Transform competent cells with plasmid (?)</li>
+
  <ul>
−
<li>10/08:Ran GoTaq colony PCR of ApPst, screened for correct product, ran gel</li>  
+
  <li>03/08:Plasmid (pBC 29 Keasling collection )loss experiment initiated</li>
−
<li>10/08:Performed a gibson assembly of EcPhoE Q5 PCR product (from 05/08)</li>
+
  <li>05/08:Q5 PCR of SmPst, EcPst, EcPhoE</li>
−
<li>10/08:Ran a gel of SmPst and EcPst Q5 products (from 05/08), then digested with Dpn1, nanodropped, PCR purified and performed a gibson assembly</li.
+
  <li>17/08:Third attempt at growth assay - did not test any Pst or PhoE genes, results ok</li>
−
<li>10/08:Ran an overhang-adding step-up PCR of KoPPK, pkDM12, pBC9, pBC29</li>
+
  <li>06/08:Inoculated overnight cultures of ApPst, ApPPK (SK-12, BA-91, UW-1)</li>
−
<li>11/08:Ran a <em>Pseudomonas aeruginosa</em> colony PCR to amplify PaOprO and PaOprP</li>
+
  <li>06/08:Ran GoTaq Colony PCR of same colonies of ApPst, ApPPK (SK-12, BA-91, UW-1)</li>
−
<li>11/08:Ran a GoTaq colony PCR of all KEIO collection genes used (growth assay team), ran gels to verify that knockouts were present</li>
+
  <li>06/08:Ran gel electrophoresis of colony PCR</li>
−
<li>11/08:Glassmilk was made</li>
+
  <li>06/08:Inoculated overnight cultures of BW25113, pkDM12, KoPPK</li>
−
<li>11/08:NMR test ran on BW25113</li>
+
  <li>17/08:Fourth attempt at growth assay - exact repeat to verify results</li>
−
<li>12/08:Another phosphate assay run- using more formic acid, not neutralised this time. </li>
+
  <li>07/08:Purified plasmids from overnight cultures of ApPst, ApPPK (SK-12, BA-91, UW-1)- "mini-prep"</li>
−
<li>12/08:Digested lacZ pSB1C3 with EcoR1, ran gel, and performed gel extraction and purification protocols</li>
+
  <li>07/08:Used Nanodrop machine to measure concentration of miniprep products</li>
−
<li>12/08:Fifth attempt at growth assay - new plate design with increased phosphate levels</li>
+
  <li>07/08:Performed an analytical digest of purified plasmids with EcoR1 and Pst1</li>
−
<li>13/08:Digest pAdapt with Sma1</li>
+
  </ul>
−
<li>13/08:Ran Gibson assembly of EcPstSCAB, SmPstSCAB, KoPPK, pKDM12, EcPPX, EcPPK "colony 1 and 2", EcPhoE</li>
+
</div>
−
<li>13/08:Transformed BW2115 with gibson products and plated on agar.</li>
+
−
<li>14/08:Ran GoTaq PCR of colonies from Gibson plates and performed gel electrophoresis with PCR products.</li>  
+
−
<li>14/08:Ran another GoTaq colony PCR of all KEIO collection genes used(growth assay team), to verify the knockouts that didn't work the first time around</li>
+
−
<li>14/08:Same phosphate assay as on the 12th but neutralised with 1M sodium hydroxide  - did not work</li>
+
−
</ul>
+
  
−
<h3>Week 9 - August 17-21  </h3>
+
<div class="DatePeriod" id="Week8">
−
<ul>
+
  <h3>Week 8 - August 10-14    </h3>
−
<li>17/08:Make liquid cultures of colonies that were verified with the gel on the 14th(EcPPX, EcPPK, pKDM12) </li>
+
  <ul>
−
<li>17/08:Attempt colony PCR of different colonies for gibsons that did not work (EcPst, KoPPK, SmPst, EcPhoE)</li>
+
  <li>10/08:Transform competent cells with plasmid (?)</li>
−
<li>17/08:PCR amplified the digest of lacZ pSB1C3 (from 12/08) to make sure that no product is in the plasmid</li>
+
  <li>10/08:Ran GoTaq colony PCR of ApPst, screened for correct product, ran gel</li>  
−
<li>17/08:Ran gel of PaOprO/PaOprP PCR attempt #1 - no positive results, even control failed</li>
+
  <li>10/08:Performed a gibson assembly of EcPhoE Q5 PCR product (from 05/08)</li>
−
<li>17/08:Glassmilk was used to isolate PolyP during yet another phosphate assay - this also did not work.</li>
+
  <li>10/08:Ran a gel of SmPst and EcPst Q5 products (from 05/08), then digested with Dpn1, nanodropped, PCR purified and performed a gibson assembly</li.
−
<li>18/08:New phosphate assay was run on PPK, PPX and BW2115 with formic acid, this time neutralised with 6M sodium hydroxide.</li>
+
  <li>10/08:Ran an overhang-adding step-up PCR of KoPPK, pkDM12, pBC9, pBC29</li>
−
<li>19/08:Growth median assay on all of KEIO collection for phosphate assay purposes</li>
+
  <li>11/08:Ran a <em>Pseudomonas aeruginosa</em> colony PCR to amplify PaOprO and PaOprP</li>
−
<li>19/08:<em>Pseudomonas aeruginosa</em> PCR attempt #2 - used more colony and made colony dilution. No positive results. Control failed.</li>
+
  <li>11/08:Ran a GoTaq colony PCR of all KEIO collection genes used (growth assay team), ran gels to verify that knockouts were present</li>
−
<li>20/08:Prepared a 3 hour and a 12 hour growth median assay. 3 hour test failed, and although the 12 hour test was completed, no results were conclusive.</li>
+
  <li>11/08:Glassmilk was made</li>
−
<li>20/08:<em>Pseudomonas aeruginosa</em> PCR attempt #3 - changed extension time from 30 to 40 seconds. No positive results. Control failed.</li>
+
  <li>11/08:NMR test ran on BW25113</li>
−
<li>20/08:Sixth attempt at growth assay - only tested PstC and PstA to see which one had an increased growth phenotype</li>
+
  <li>12/08:Another phosphate assay run- using more formic acid, not neutralised this time. </li>
−
<li>20/08:<em>Pseudomonas aeruginosa</em> PCR attempt #4 - used 3 differnet polymerases- Taq, Q5, Phusion. Correct bands visible only with Taq.</li>
+
  <li>12/08:Digested lacZ pSB1C3 with EcoR1, ran gel, and performed gel extraction and purification protocols</li>
−
<li>20/08:Gibson of pAdapt digested with Sma1 and ApPst 1 and 2</li>
+
  <li>12/08:Fifth attempt at growth assay - new plate design with increased phosphate levels</li>
−
<li>21/08:12 hour growth median assay ran - absorbances were too high, a precipitate formed </li>
+
  <li>13/08:Digest pAdapt with Sma1</li>
−
</ul>
+
  <li>13/08:Ran Gibson assembly of EcPstSCAB, SmPstSCAB, KoPPK, pKDM12, EcPPX, EcPPK "colony 1 and 2", EcPhoE</li>
 +
  <li>13/08:Transformed BW2115 with gibson products and plated on agar.</li>
 +
  <li>14/08:Ran GoTaq PCR of colonies from Gibson plates and performed gel electrophoresis with PCR products.</li>  
 +
  <li>14/08:Ran another GoTaq colony PCR of all KEIO collection genes used(growth assay team), to verify the knockouts that didn't work the first time around</li>
 +
  <li>14/08:Same phosphate assay as on the 12th but neutralised with 1M sodium hydroxide  - did not work</li>
 +
  </ul>
 +
</div>
  
−
<h3>Week 10 - August 24-28    </h3>
+
<div class="DatePeriod" id="Week9">
−
<ul>
+
  <h3>Week 9 - August 17-21  </h3>
−
<li>24/08:Run double digest the gibson assembled plasmids (EcPPX, EcPPK, pKDM12, KoPPK) with Xba1 and Xba1 + Spe1</li>
+
  <ul>
−
<li>25/08:Nanodrop lacZ #1,2,3 and transform BW2115 with lacZ #1 plasmid, plate on X-Gal/IPTG agar for blue-white screening</li>
+
  <li>17/08:Make liquid cultures of colonies that were verified with the gel on the 14th(EcPPX, EcPPK, pKDM12) </li>
−
<li>26/08:GoTaq PCR screen of EcPst, EcPhoE, SmPst, ApPst</li>
+
  <li>17/08:Attempt colony PCR of different colonies for gibsons that did not work (EcPst, KoPPK, SmPst, EcPhoE)</li>
−
<li>26/08:<em>Pseudomonas aeruginosa</em> PCR attempt #5 - new Phusion polymerase and buffer used- still no positive results. Control failed.</li>
+
  <li>17/08:PCR amplified the digest of lacZ pSB1C3 (from 12/08) to make sure that no product is in the plasmid</li>
−
<li>26/08:Formic acid phosphate assay ran on PPK, PPX and BW25113. </li>
+
  <li>17/08:Ran gel of PaOprO/PaOprP PCR attempt #1 - no positive results, even control failed</li>
−
<li>26/08:Double digest of EcPPK, EcPPx, KoPPK, pKDM12 with Xba1 and Spe1, ran gel electrophoresis</li>
+
  <li>17/08:Glassmilk was used to isolate PolyP during yet another phosphate assay - this also did not work.</li>
−
<li>27/08:<em>Pseudomonas aeruginosa</em> PCR attempt #6 - changed extension time to 35 seconds, raised annealing temperature from 60 to 65 degrees- still no positive results. Control failed.</li>
+
  <li>18/08:New phosphate assay was run on PPK, PPX and BW2115 with formic acid, this time neutralised with 6M sodium hydroxide.</li>
−
<li>27/08:Competent cells made for phosphate assay</li>
+
  <li>19/08:Growth median assay on all of KEIO collection for phosphate assay purposes</li>
−
<li>27/08:Made grid plates of EcPhoE, EcPst and SmPst to screen colonies that have been transformed with the respective plasmid</li>
+
  <li>19/08:<em>Pseudomonas aeruginosa</em> PCR attempt #2 - used more colony and made colony dilution. No positive results. Control failed.</li>
−
<img src="https://static.igem.org/mediawiki/2015/thumb/1/10/EcPhoE_Screen_Plate.jpg/665px-EcPhoE_Screen_Plate.jpg" height="25%" width="25%"/>
+
  <li>20/08:Prepared a 3 hour and a 12 hour growth median assay. 3 hour test failed, and although the 12 hour test was completed, no results were conclusive.</li>
−
<img src="https://static.igem.org/mediawiki/2015/thumb/4/47/EcPST_Screen_Plate.jpg/675px-EcPST_Screen_Plate.jpg"height="25%" width="25% "/>
+
  <li>20/08:<em>Pseudomonas aeruginosa</em> PCR attempt #3 - changed extension time from 30 to 40 seconds. No positive results. Control failed.</li>
−
<img src="https://static.igem.org/mediawiki/2015/thumb/1/1b/ApPst_Screen_Plate.jpg/600px-ApPst_Screen_Plate.jpg"height="25%" width="25% "/>
+
  <li>20/08:Sixth attempt at growth assay - only tested PstC and PstA to see which one had an increased growth phenotype</li>
−
</ul>
+
  <li>20/08:<em>Pseudomonas aeruginosa</em> PCR attempt #4 - used 3 differnet polymerases- Taq, Q5, Phusion. Correct bands visible only with Taq.</li>
 +
  <li>20/08:Gibson of pAdapt digested with Sma1 and ApPst 1 and 2</li>
 +
  <li>21/08:12 hour growth median assay ran - absorbances were too high, a precipitate formed </li>
 +
  </ul>
 +
</div>
  
 +
<div class="DatePeriod" id="Week10">
 +
  <h3>Week 10 - August 24-28    </h3>
 +
  <ul>
 +
  <li>24/08:Run double digest the gibson assembled plasmids (EcPPX, EcPPK, pKDM12, KoPPK) with Xba1 and Xba1 + Spe1</li>
 +
  <li>25/08:Nanodrop lacZ #1,2,3 and transform BW2115 with lacZ #1 plasmid, plate on X-Gal/IPTG agar for blue-white screening</li>
 +
  <li>26/08:GoTaq PCR screen of EcPst, EcPhoE, SmPst, ApPst</li>
 +
  <li>26/08:<em>Pseudomonas aeruginosa</em> PCR attempt #5 - new Phusion polymerase and buffer used- still no positive results. Control failed.</li>
 +
  <li>26/08:Formic acid phosphate assay ran on PPK, PPX and BW25113. </li>
 +
  <li>26/08:Double digest of EcPPK, EcPPx, KoPPK, pKDM12 with Xba1 and Spe1, ran gel electrophoresis</li>
 +
  <li>27/08:<em>Pseudomonas aeruginosa</em> PCR attempt #6 - changed extension time to 35 seconds, raised annealing temperature from 60 to 65 degrees- still no positive results. Control failed.</li>
 +
  <li>27/08:Competent cells made for phosphate assay</li>
 +
  <li>27/08:Made grid plates of EcPhoE, EcPst and SmPst to screen colonies that have been transformed with the respective plasmid</li>
 +
  <img src="https://static.igem.org/mediawiki/2015/thumb/1/10/EcPhoE_Screen_Plate.jpg/665px-EcPhoE_Screen_Plate.jpg" height="25%" width="25%"/>
 +
  <img src="https://static.igem.org/mediawiki/2015/thumb/4/47/EcPST_Screen_Plate.jpg/675px-EcPST_Screen_Plate.jpg"height="25%" width="25% "/>
 +
  <img src="https://static.igem.org/mediawiki/2015/thumb/1/1b/ApPst_Screen_Plate.jpg/600px-ApPst_Screen_Plate.jpg"height="25%" width="25% "/>
 +
  </ul>
 +
</div>
  
−
<h3>Week 11 - August 31- September 4    </h3>
+
<div class="DatePeriod" id="Week11">
−
<ul>
+
  <h3>Week 11 - August 31- September 4    </h3>
−
<li>28/08:2nd attempt at double digest of EcPPK, EcPPx, KoPPK, pKDM12 with Xba1 and Spe1 to get clearer gel results</li>
+
  <ul>
−
<img src="https://static.igem.org/mediawiki/2015/3/34/Ecppx_KoPPK_digests-XbaI.SpeI_.PNG" height="25%" width="25%"/>
+
  <li>28/08:2nd attempt at double digest of EcPPK, EcPPx, KoPPK, pKDM12 with Xba1 and Spe1 to get clearer gel results</li>
−
<img src="https://static.igem.org/mediawiki/2015/thumb/3/3f/EcPPK_pKDM12-XbaI.Spe1.PNG/600px-EcPPK_pKDM12-XbaI.Spe1.PNG" height="25%" width="25%"/>
+
  <img src="https://static.igem.org/mediawiki/2015/3/34/Ecppx_KoPPK_digests-XbaI.SpeI_.PNG" height="25%" width="25%"/>
−
<li>03/09:Tests to see if fluorimetry is a viable means to measure polyphosphate in the cells</li>
+
  <img src="https://static.igem.org/mediawiki/2015/thumb/3/3f/EcPPK_pKDM12-XbaI.Spe1.PNG/600px-EcPPK_pKDM12-XbaI.Spe1.PNG" height="25%" width="25%"/>
−
<li>03/09:Slides for fluorescence microscopy prepared using DAPI staining</li>
+
  <li>03/09:Tests to see if fluorimetry is a viable means to measure polyphosphate in the cells</li>
−
<li>04/09:Microscopy reveals polyphosphate chains are visible - success!! </li>
+
  <li>03/09:Slides for fluorescence microscopy prepared using DAPI staining</li>
−
<li>04/09:Miniprep of ApPst colonies #22, 23, 24, 37, 38, 39. (tested concentraions with nanodrop)</li>
+
  <li>04/09:Microscopy reveals polyphosphate chains are visible - success!! </li>
−
<li>04/09:Double digest of mini-prepped ApPst with EcoR1 and Pst1</li>
+
  <li>04/09:Miniprep of ApPst colonies #22, 23, 24, 37, 38, 39. (tested concentraions with nanodrop)</li>
−
<li>04/09:Yet another phosphate assay with formic acid done (same strains) - decent results </li>
+
  <li>04/09:Double digest of mini-prepped ApPst with EcoR1 and Pst1</li>
−
</ul>
+
  <li>04/09:Yet another phosphate assay with formic acid done (same strains) - decent results </li>
 +
  </ul>
 +
</div>
  
−
<h3>Week 12 - September 7-11  </h3>
+
<div class="DatePeriod" id="Week12">
−
<ul>
+
  <h3>Week 12 - September 7-11  </h3>
−
<li>07/09:Phosphate assay team analysed water samples from around the world- Israel, Egypt...</li>
+
  <ul>
−
<li>07/09:<em>Pseudomonas aeruginosa</em> PCR attempt #7 - fresh plate of bacteria used- research showed that a biofil forms on the bacteria and prevents proper amplification- still no positive results. Control failed.</li>
+
  <li>07/09:Phosphate assay team analysed water samples from around the world- Israel, Egypt...</li>
−
<li>08/09:Colony PCR on competent cells transformed with PstC and PPK</li>
+
  <li>07/09:<em>Pseudomonas aeruginosa</em> PCR attempt #7 - fresh plate of bacteria used- research showed that a biofil forms on the bacteria and prevents proper amplification- still no positive results. Control failed.</li>
−
</ul>
+
  <li>08/09:Colony PCR on competent cells transformed with PstC and PPK</li>
 +
  </ul>
 +
</div>
  
−
<h3>Week 13 - September 13-18  </h3>
+
<div class="DatePeriod" id="Week13">
−
<ul>
+
  <h3>Week 13 - September 13-18  </h3>
−
</ul>
+
  <ul>
 +
  </ul>
 +
</div>
 +
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Revision as of 13:23, 9 September 2015


Notebook

The following is a weekly description of the experiments carried out each week. To see the protocols click here

  • Dry Lab Work Period
  • Week 1
  • Week 2
  • Week 3
  • Week 4
  • Week 5
  • Week 6
  • Week 7
  • Week 8
  • Week 9
  • Week 10
  • Week 11
  • Week 12
  • Week 13

Dry Lab Work Period

  • 28/01: Brief introductory meeting about the iGEM competition
  • 17/03: The official iGEM team to represent the University of York was formed!
  • Daily dry lab research begins, primer and construct designs made and ordered.
  • 05/05: Articlepublished in Nouse (University newspaper) about the 2015 iGEM team.
  • 03/06: We were invited to Science and Technology Alumni Network discussion event (outreach)
  • 04/06: Articlepublished on the University's central news about this years iGEM team.
  • 12/06: A meeting was held with Yorkshire Water to discuss water remediation processes in the Yorkshire region
  • 13/06: We were invited to a University of York Biology Alumni event
  • 15/06:
    1. Some of us went to a round-table discussion about the impact of Santander at York as some of the team members won the International Connections Award by Santander.
    2. Article published on Mind the Horizon about University of York iGEM
  • 19/06:
    1. Lab safety induction was carried out
    2. We passed out pamphlets at the York Festival of Ideas to raise awareness about the iGEM competition and synthetic biology.
  • 26&27/06: We participated in giving talks to prospective students about iGEM.

Week 1 - June 23-27

  • 24/07: Wet lab practice day- reviewed basic lab protocols- making LB, agar plates, competent cells, transformations, PCR

Week 2 - June 29- July 3

  • 01/07:Competent cells made
  • 02/07:Plates were streaked with two isolates of each deletion wanted from the Keio collection [PPX, PPK, PstA, PstB, PstC, PstS and PhoE] These have been collected and put in the incubating (37°C) room.
  • 03/07:Competent cells were tested with iGEM transformation efficiency kit

Week 3 - July 6-10

  • 07/07: More agar plates made, next set of competent cell procedure started
  • 07/07: YUfund page done and submitted

Week 4 - July 13-17

  • 14/07: Vistited Badger Hill Primary School and held a bacteria workshop for the students
  • 15/07: first day of demonstrations of cell transformations and aseptic technique to sixth formers
  • 16/07: second day of demonstrations to sixth formers:analysing transformations and building bioreactors
  • 17/07: analysing bioreactors with sixth formers
  • 17/07: phosphate assay done to get a standard curve
  • 17/07: first attempt at growth assay - had to be redone, as computer crashed and updated mid-run

Week 5 - July 20-24

  • 20/07:Phosphate assay was started for KEIO collection - PPK, PPX, wildtype - no results
  • 20/07:Colony PCR on SmPstSCAB, EcPstSCAB and EcPhoE
  • 20/07:Next set of competent cells made
  • 22/07:X-Gal/IPTG Plates made
  • 22/07:Digested pSB1C3 with EcoRI and DpnI, then used Gibson Assembly to insert our adapter genes in.
  • 23/07:Visit to AgeUK LunchClub to explain about the GM
  • 23/07:Glycerol stocks of Sinorhizobium meliloti
  • 23/07:Phosphate assay of PPK, PPX, wildtype again with formic acid. Samples were neutralised before plating. - no results
  • 24/07:Ran gel electrophoresis of PCR from 22/07
  • 24/07:Second attempt at growth assay - had to be redone due to poor cell growth, potentially caused by poor spreading of plates

Week 6 - July 27-31

  • 27/07:Preparations for glassmilk procedure
  • 28/07:First day of outreaching event to Saint Helen's CHurch, York, to carry out strawberry extraction practical and briefly explain about the steps involved for GM
  • 29/07:Second (and the last day) of outreaching event to Saint Helen's Church, York, to carry out strawberry extraction practical and briefly explain about the steps involved for GM
  • 30/07:After several repeats the phosphate assay team managed to get significant data
  • 30/07:Phosphate assay on Keasling strains: pBC9, pBC29 and pKDM12.

Week 7 - August 3-7

  • 03/08:Plasmid (pBC 29 Keasling collection )loss experiment initiated
  • 05/08:Q5 PCR of SmPst, EcPst, EcPhoE
  • 17/08:Third attempt at growth assay - did not test any Pst or PhoE genes, results ok
  • 06/08:Inoculated overnight cultures of ApPst, ApPPK (SK-12, BA-91, UW-1)
  • 06/08:Ran GoTaq Colony PCR of same colonies of ApPst, ApPPK (SK-12, BA-91, UW-1)
  • 06/08:Ran gel electrophoresis of colony PCR
  • 06/08:Inoculated overnight cultures of BW25113, pkDM12, KoPPK
  • 17/08:Fourth attempt at growth assay - exact repeat to verify results
  • 07/08:Purified plasmids from overnight cultures of ApPst, ApPPK (SK-12, BA-91, UW-1)- "mini-prep"
  • 07/08:Used Nanodrop machine to measure concentration of miniprep products
  • 07/08:Performed an analytical digest of purified plasmids with EcoR1 and Pst1

Week 8 - August 10-14

  • 10/08:Transform competent cells with plasmid (?)
  • 10/08:Ran GoTaq colony PCR of ApPst, screened for correct product, ran gel
  • 10/08:Performed a gibson assembly of EcPhoE Q5 PCR product (from 05/08)
  • 10/08:Ran a gel of SmPst and EcPst Q5 products (from 05/08), then digested with Dpn1, nanodropped, PCR purified and performed a gibson assembly10/08:Ran an overhang-adding step-up PCR of KoPPK, pkDM12, pBC9, pBC29
  • 11/08:Ran a Pseudomonas aeruginosa colony PCR to amplify PaOprO and PaOprP
  • 11/08:Ran a GoTaq colony PCR of all KEIO collection genes used (growth assay team), ran gels to verify that knockouts were present
  • 11/08:Glassmilk was made
  • 11/08:NMR test ran on BW25113
  • 12/08:Another phosphate assay run- using more formic acid, not neutralised this time.
  • 12/08:Digested lacZ pSB1C3 with EcoR1, ran gel, and performed gel extraction and purification protocols
  • 12/08:Fifth attempt at growth assay - new plate design with increased phosphate levels
  • 13/08:Digest pAdapt with Sma1
  • 13/08:Ran Gibson assembly of EcPstSCAB, SmPstSCAB, KoPPK, pKDM12, EcPPX, EcPPK "colony 1 and 2", EcPhoE
  • 13/08:Transformed BW2115 with gibson products and plated on agar.
  • 14/08:Ran GoTaq PCR of colonies from Gibson plates and performed gel electrophoresis with PCR products.
  • 14/08:Ran another GoTaq colony PCR of all KEIO collection genes used(growth assay team), to verify the knockouts that didn't work the first time around
  • 14/08:Same phosphate assay as on the 12th but neutralised with 1M sodium hydroxide - did not work

Week 9 - August 17-21

  • 17/08:Make liquid cultures of colonies that were verified with the gel on the 14th(EcPPX, EcPPK, pKDM12)
  • 17/08:Attempt colony PCR of different colonies for gibsons that did not work (EcPst, KoPPK, SmPst, EcPhoE)
  • 17/08:PCR amplified the digest of lacZ pSB1C3 (from 12/08) to make sure that no product is in the plasmid
  • 17/08:Ran gel of PaOprO/PaOprP PCR attempt #1 - no positive results, even control failed
  • 17/08:Glassmilk was used to isolate PolyP during yet another phosphate assay - this also did not work.
  • 18/08:New phosphate assay was run on PPK, PPX and BW2115 with formic acid, this time neutralised with 6M sodium hydroxide.
  • 19/08:Growth median assay on all of KEIO collection for phosphate assay purposes
  • 19/08:Pseudomonas aeruginosa PCR attempt #2 - used more colony and made colony dilution. No positive results. Control failed.
  • 20/08:Prepared a 3 hour and a 12 hour growth median assay. 3 hour test failed, and although the 12 hour test was completed, no results were conclusive.
  • 20/08:Pseudomonas aeruginosa PCR attempt #3 - changed extension time from 30 to 40 seconds. No positive results. Control failed.
  • 20/08:Sixth attempt at growth assay - only tested PstC and PstA to see which one had an increased growth phenotype
  • 20/08:Pseudomonas aeruginosa PCR attempt #4 - used 3 differnet polymerases- Taq, Q5, Phusion. Correct bands visible only with Taq.
  • 20/08:Gibson of pAdapt digested with Sma1 and ApPst 1 and 2
  • 21/08:12 hour growth median assay ran - absorbances were too high, a precipitate formed

Week 10 - August 24-28

  • 24/08:Run double digest the gibson assembled plasmids (EcPPX, EcPPK, pKDM12, KoPPK) with Xba1 and Xba1 + Spe1
  • 25/08:Nanodrop lacZ #1,2,3 and transform BW2115 with lacZ #1 plasmid, plate on X-Gal/IPTG agar for blue-white screening
  • 26/08:GoTaq PCR screen of EcPst, EcPhoE, SmPst, ApPst
  • 26/08:Pseudomonas aeruginosa PCR attempt #5 - new Phusion polymerase and buffer used- still no positive results. Control failed.
  • 26/08:Formic acid phosphate assay ran on PPK, PPX and BW25113.
  • 26/08:Double digest of EcPPK, EcPPx, KoPPK, pKDM12 with Xba1 and Spe1, ran gel electrophoresis
  • 27/08:Pseudomonas aeruginosa PCR attempt #6 - changed extension time to 35 seconds, raised annealing temperature from 60 to 65 degrees- still no positive results. Control failed.
  • 27/08:Competent cells made for phosphate assay
  • 27/08:Made grid plates of EcPhoE, EcPst and SmPst to screen colonies that have been transformed with the respective plasmid

Week 11 - August 31- September 4

  • 28/08:2nd attempt at double digest of EcPPK, EcPPx, KoPPK, pKDM12 with Xba1 and Spe1 to get clearer gel results
  • 03/09:Tests to see if fluorimetry is a viable means to measure polyphosphate in the cells
  • 03/09:Slides for fluorescence microscopy prepared using DAPI staining
  • 04/09:Microscopy reveals polyphosphate chains are visible - success!!
  • 04/09:Miniprep of ApPst colonies #22, 23, 24, 37, 38, 39. (tested concentraions with nanodrop)
  • 04/09:Double digest of mini-prepped ApPst with EcoR1 and Pst1
  • 04/09:Yet another phosphate assay with formic acid done (same strains) - decent results

Week 12 - September 7-11

  • 07/09:Phosphate assay team analysed water samples from around the world- Israel, Egypt...
  • 07/09:Pseudomonas aeruginosa PCR attempt #7 - fresh plate of bacteria used- research showed that a biofil forms on the bacteria and prevents proper amplification- still no positive results. Control failed.
  • 08/09:Colony PCR on competent cells transformed with PstC and PPK

Week 13 - September 13-18