Difference between revisions of "Team:York/Test"

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<body>
 
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<div class="col-md-1"></div>
 
<div class="col-md-1"></div>
 
<div class="col-md-10">
 
<div class="col-md-10">
 +
<!-- CONTENT GOES HERE :D -->
 +
 +
  <h1>Notebook</h1>
 +
<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>
  
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    <center>
 
−
        <h1 style="font-family:Garamond"> Protocols </h1>
 
−
    </center>
 
−
   
 
 
<div class="col-md-3">
 
<div class="col-md-3">
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    <div id="wrap">
+
<div class="navbar">
−
        <ul class="navbar">
+
    <ul>
−
            <li>
+
        <li style="display:block;" onclick="toggleWeek('DryLab')">Dry Lab Work Period</li>
−
                <a href="https://2015.igem.org/Team:York/Protocols/Growth_Media">Growth Media</a>
+
        <li style="display:block;" onclick="toggleWeek('Week1')">Week 1</li>
−
            </li>
+
        <li style="display:block;" onclick="toggleWeek('Week2')">Week 2</li>
−
            <li>
+
        <li style="display:block;" onclick="toggleWeek('Week3')">Week 3</li>
−
                <a href="https://2015.igem.org/wiki/index.php?title=Team:York/Protocols/CompetentCells">Competent Cells</a>
+
        <li style="display:block;" onclick="toggleWeek('Week4')">Week 4</li>
−
            </li>
+
        <li style="display:block;" onclick="toggleWeek('Week5')">Week 5</li>
−
            <li>
+
        <li style="display:block;" onclick="toggleWeek('Week6')">Week 6</li>
−
                <a href="#">Transformations</a>
+
        <li style="display:block;" onclick="toggleWeek('Week7')">Week 7</li>
−
                <ul>
+
        <li style="display:block;" onclick="toggleWeek('Week8')">Week 8</li>
−
                    <li>
+
        <li style="display:block;" onclick="toggleWeek('Week9')">Week 9</li>
−
                        <a href="#" >Transformation Efficiency Tests</a>
+
        <li style="display:block;" onclick="toggleWeek('Week10')">Week 10</li>
−
                    </li>
+
        <li style="display:block;" onclick="toggleWeek('Week11')">Week 11</li>
−
                    <li>
+
        <li style="display:block;" onclick="toggleWeek('Week12')">Week 12</li>
−
                        <a href="#">Plasmids</a>
+
        <li style="display:block;" onclick="toggleWeek('Week13')">Week 13</li>
−
                    </li>
+
    </ul>
−
                 
+
</div>
−
                </ul>
+
−
            </li>
+
−
            <li>
+
−
                <a href="https://2015.igem.org/Team:York/Protocols/PCR">PCR</a>
+
−
                <ul>
+
−
                    <li>
+
−
                        <a href="#">General</a>
+
−
                    </li>
+
−
                   
+
−
                </ul>
+
−
            </li>
+
−
            <li>
+
−
                <a href="#">Gel Extractions</a>
+
−
               
+
−
            </li>
+
−
            <li>
+
−
                <a href="https://2015.igem.org/Team:York/Protocols/PhosphateAssay">Phosphate Assay</a>
+
−
                <ul>
+
−
                    <li>
+
−
                        <a href="https://2015.igem.org/Team:York/Protocols/PhosphateAssay_Glassmilk">Glassmilk</a>
+
−
                   
+
−
                </ul>
+
−
            </li>
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−
        </ul>
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    </div>
+
 
</div>
 
</div>
  
 
<div class="col-md-9">
 
<div class="col-md-9">
−
<h2>Lysogeny Broth</h2>
+
<div class="DatePeriod" id="DryLab">
 +
  <h3>Dry Lab Work Period</h3>
 +
  <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>Materials</h3>
+
<div class="DatePeriod" id="Week1">
−
<ul>
+
  <h3>Week 1 - June 23-27 </h3>
−
<li>10g of tryptone</li>
+
  <ul>
−
<li>5g of yeast extract</li>
+
  <li>24/07: Wet lab practice day- reviewed basic lab protocols- making LB, agar plates, competent cells, transformations, PCR </li>
−
<li>10g of NaCl</li>
+
  </ul>
−
<li>1L of Deionized Water</li>
+
</div>
−
<li> 1M NaCl </li>
+
−
<li> 1M KOH </li>
+
−
</ul>
+
  
−
<h3>Procedure</h3>
+
<div class="DatePeriod" id="Week2">
−
<ol>
+
  <h3>Week 2 - June 29- July 3 </h3>
−
<li>Use a container with at least double the volume of the liquid that you are making.</li>
+
  <ul>
−
<li>Dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 950 mL deionized water. </li>
+
  <li>01/07:Competent cells made</li>
−
<li>Adjust the pH of the medium to 7.0 using 1M NaOH or KOH and bring volume up to 1 liter. </li>
+
  <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>Autoclave. </li>
+
  <li>03/07:Competent cells were tested with iGEM transformation efficiency kit</li>
−
<li>Store at room temperature or +4°C.</li>
+
  </ul>
−
</ol>
+
</div>
  
−
<h2>LB Agar</h2>
+
<div class="DatePeriod" id="Week3">
−
<h4>Procedure</h4>
+
  <h3>Week 3 - July 6-10 </h3>
−
<ol>
+
  <ul>
−
<li>follow steps to make lysogeny broth as above</li>
+
  <li>07/07: More agar plates made, next set of competent cell procedure started</li>
−
<li>add 15g of agar powder per litre of LB</li>
+
  <li>07/07: YUfund page done and submitted</li>
−
<li>autoclave in 200 or 250 mL aliquots in 500mL duran bottles</li>
+
  </ul>
−
<li>add antibiotic desired to melted agar (~55°C)</li>
+
</div>
−
<li>pour into petri dishes and leave to harden in asceptic fume hood</li>
+
−
<li>invert, label, wrap with parafilm and store at 4°C</li>
+
−
</ol>
+
  
−
<h2>X-Gal/IPTG Protocol</h2>
+
<div class="DatePeriod" id="Week4">
−
<p>add the following to 200 mL of melted (~55°C) LB-Agar</p>
+
  <h3>Week 4 - July 13-17  </h3>
−
<ul><li>20 uL X-Gal (20mg/ml)</li>
+
  <ul>
−
<li>20 uL IPTG (100mM)</li>
+
  <li>14/07: Vistited Badger Hill Primary School and held a bacteria workshop for the students</li>
−
<li>20 uL of appropriate antibiotic</li>
+
  <li>15/07: first day of demonstrations of cell transformations and aseptic technique to sixth formers</li>
−
</ul>
+
  <li>16/07: second day of demonstrations to sixth formers:analysing transformations and building bioreactors</li>
−
<p>swirl to mix, try to avoid making bubbles</p>
+
  <Li>17/07: analysing bioreactors with sixth formers</li>
−
<p>dry at room temp and wrap in foil to prevent degradation</p>
+
  <li>17/07: phosphate assay done to get a standard curve</li>
 +
  <li>17/07: first attempt at growth assay - had to be redone, as computer crashed and updated mid-run</li>
 +
  </ul>
 +
</div>
  
 +
<div class="DatePeriod" id="Week5">
 +
  <h3>Week 5 - July 20-24  </h3>
 +
  <ul>
 +
  <li>20/07:Phosphate assay was started for KEIO collection - PPK, PPX, wildtype - no results</li>
 +
  <li>20/07:Colony PCR on SmPstSCAB, EcPstSCAB and EcPhoE</li>
 +
  <li>20/07:Next set of competent cells made </li>
 +
  <li>22/07:X-Gal/IPTG Plates made </li>
 +
  <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>
  
−
<h2>10X TAE (Tris Acetate EDTA) Buffer</h2>
+
<div class="DatePeriod" id="Week6">
−
<ul>Dissolve the following in 600mL of dH<sub>2</sub>O</ul>
+
  <h3>Week 6 - July 27-31  </h3>
−
<ol><li> 48.4 g Tris base (FW 121)</li>
+
  <ul>
−
<li>11.42 mL glacial acetic acid</li>
+
  <li>27/07:Preparations for glassmilk procedure</li>
−
<li>40 mL of 0.25M EDTA (pH 8.0) </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>
−
</ol></ul>
+
  <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>
−
<ul>Make up volume to 1.0 L with dH<sub>2</sub>O</ul>
+
  <li>30/07:After several repeats the phosphate assay team managed to get significant data</li>
−
<ul>Autoclave in appropriately sized bottle </ul>
+
  <li>30/07:Phosphate assay on Keasling strains: pBC9, pBC29 and pKDM12.</li>
 +
  </ul>
 +
</div>
  
−
<h2>Agarose Gel Electrophoresis</h3>
+
<div class="DatePeriod" id="Week7">
−
<ol>
+
  <h3>Week 7 - August 3-7  </h3>
−
<li>add 1g of agarose per 100 mL of TAE (1X) buffer</li>
+
  <ul>
−
<li>microwave to dissolve</li>
+
  <li>03/08:Plasmid (pBC 29 Keasling collection )loss experiment initiated</li>
−
<li>add 10 uL of Syber-Safe per 100 mL of TAE buffer</li>
+
  <li>05/08:Q5 PCR of SmPst, EcPst, EcPhoE</li>
−
<li>pour into gel tray and allow to set (don't forget the comb!) </li>
+
  <li>17/08:Third attempt at growth assay - did not test any Pst or PhoE genes, results ok</li>
−
<li>put gel into the tank and fill tank to cover top of gel </li>
+
  <li>06/08:Inoculated overnight cultures of ApPst, ApPPK (SK-12, BA-91, UW-1)</li>
−
<li>load ladder and samples and run! </li>
+
  <li>06/08:Ran GoTaq Colony PCR of same colonies of ApPst, ApPPK (SK-12, BA-91, UW-1)</li>
−
</ol>
+
  <li>06/08:Ran gel electrophoresis of colony PCR</li>
 +
  <li>06/08:Inoculated overnight cultures of BW25113, pkDM12, KoPPK</li>
 +
  <li>17/08:Fourth attempt at growth assay - exact repeat to verify results</li>
 +
  <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>
 +
</div>
  
−
<h2>Competent Cells</h2>
+
<div class="DatePeriod" id="Week8">
−
<h3>Materials</h3>
+
  <h3>Week 8 - August 10-14    </h3>
−
<ul>
+
  <ul>
−
<li>LB media (in 50 mL in 250 conical flask) pre- autoclaved (x2)</li>
+
  <li>10/08:Transform competent cells with plasmid (?)</li>
−
<li>overnight culture of DH5α (x2)</li>
+
  <li>10/08:Ran GoTaq colony PCR of ApPst, screened for correct product, ran gel</li>
−
<li>shaker in 37°C room</li>
+
  <li>10/08:Performed a gibson assembly of EcPhoE Q5 PCR product (from 05/08)</li>
−
<li>15 mL falcon tubes (~x15)</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>eppendorf tubes (1 mL)</li>
+
  <li>10/08:Ran an overhang-adding step-up PCR of KoPPK, pkDM12, pBC9, pBC29</li>
−
<li>50% glycerol</li>
+
  <li>11/08:Ran a <em>Pseudomonas aeruginosa</em> colony PCR to amplify PaOprO and PaOprP</li>
−
<li>0.1 M CaCl2 </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>waste bucket with Vircon (kill bacteria)</li>
+
  <li>11/08:Glassmilk was made</li>
−
<li>ice buckets</li>
+
  <li>11/08:NMR test ran on BW25113</li>
−
</ul>
+
  <li>12/08:Another phosphate assay run- using more formic acid, not neutralised this time. </li>
−
<h3>Protocol:</h3>
+
  <li>12/08:Digested lacZ pSB1C3 with EcoR1, ran gel, and performed gel extraction and purification protocols</li>
−
<ol>
+
  <li>12/08:Fifth attempt at growth assay - new plate design with increased phosphate levels</li>
−
<li>inoculate 1% inoculum from overnight culture</li>
+
  <li>13/08:Digest pAdapt with Sma1</li>
−
<li>1 mL  for 100 mL medium</li>
+
  <li>13/08:Ran Gibson assembly of EcPstSCAB, SmPstSCAB, KoPPK, pKDM12, EcPPX, EcPPK "colony 1 and 2", EcPhoE</li>
−
<li>2 separate 250 mL flasks each with 50 mL LB</li>
+
  <li>13/08:Transformed BW2115 with gibson products and plated on agar.</li>
−
<li>0.5 mL of culture- grow with shaking until ~ 0.375 OD (at 600 nm) - we went a bit over, but other protocols say this is ok</li>
+
  <li>14/08:Ran GoTaq PCR of colonies from Gibson plates and performed gel electrophoresis with PCR products.</li>
−
<li>put flasks on ice for 10 minutes</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>put 10 mL of inoculum into 15 mL falcon tubes (repeat to use up all the inoculum)</li>
+
  <li>14/08:Same phosphate assay as on the 12th but neutralised with 1M sodium hydroxide  - did not work</li>
−
<li>centrifuge @ 5K for 10 minutes @ 4°C</li>
+
  </ul>
−
<li>discard supernatant and resuspend pellet in 2 mL of 0.1 M CaCl2</li>
+
</div>
−
<li>chill on ice for 20 mins </li>
+
−
<li>centrifuge @ 5K for 10 minutes @ 4°C</li>
+
−
<li>discard supernatant and resuspend each pellet in 0.28 mL of 0.1 M CaCl2 and 0.12 mL of 50% glycerol - you can also prepare a solution of CaCl2 and glycerol before hand</li>
+
−
<li>aliquot 100 μL of resuspension into sterile eppendorfs</li>
+
−
<li>store at -80°C</li>
+
  
−
<h2> Colony PCR Protocols</h2>
+
<div class="DatePeriod" id="Week9">
−
<h4>For PCR reactions up to 50 μL add the following to each PCR Tube: </h4>
+
  <h3>Week 9 - August 17-21  </h3>
−
<table>
+
  <ul>
−
                <tr>
+
  <li>17/08:Make liquid cultures of colonies that were verified with the gel on the 14th(EcPPX, EcPPK, pKDM12) </li>
−
                    <td>Substance</td>
+
  <li>17/08:Attempt colony PCR of different colonies for gibsons that did not work (EcPst, KoPPK, SmPst, EcPhoE)</li>
−
                    <td>GoTaq</td>
+
  <li>17/08:PCR amplified the digest of lacZ pSB1C3 (from 12/08) to make sure that no product is in the plasmid</li>
−
                    <td>Q5</td>
+
  <li>17/08:Ran gel of PaOprO/PaOprP PCR attempt #1 - no positive results, even control failed</li>
−
                    <td>Phusion</td>
+
  <li>17/08:Glassmilk was used to isolate PolyP during yet another phosphate assay - this also did not work.</li>
−
                </tr>
+
  <li>18/08:New phosphate assay was run on PPK, PPX and BW2115 with formic acid, this time neutralised with 6M sodium hydroxide.</li>
−
                <tr>
+
  <li>19/08:Growth median assay on all of KEIO collection for phosphate assay purposes</li>
−
                    <td>Forward Primer</td>
+
  <li>19/08:<em>Pseudomonas aeruginosa</em> PCR attempt #2 - used more colony and made colony dilution. No positive results. Control failed.</li>
−
                    <td>2.5</td>
+
  <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>
−
                    <td>2.5</td>
+
  <li>20/08:<em>Pseudomonas aeruginosa</em> PCR attempt #3 - changed extension time from 30 to 40 seconds. No positive results. Control failed.</li>
−
                    <td>2.5</td>
+
   <li>20/08:Sixth attempt at growth assay - only tested PstC and PstA to see which one had an increased growth phenotype</li>
−
              </tr>
+
   <li>20/08:<em>Pseudomonas aeruginosa</em> PCR attempt #4 - used 3 differnet polymerases- Taq, Q5, Phusion. Correct bands visible only with Taq.</li>
−
              <tr>
+
   <li>20/08:Gibson of pAdapt digested with Sma1 and ApPst 1 and 2</li>
−
                    <td>Reverse Primer</td>
+
  <li>21/08:12 hour growth median assay ran - absorbances were too high, a precipitate formed </li>
−
                    <td>2.5</td>
+
   </ul>
−
                    <td>2.5</td>
+
</div>
−
                    <td>2.5</td>
+
−
              </tr>
+
−
              <tr>
+
−
                    <td>Polmerase</td>  
+
−
                    <td>.2</td>  
+
−
                    <td>25 μL mastermix</td>
+
−
                    <td>.5</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>dNTPs</td>
+
−
                    <td>.5</td>
+
−
                    <td>-</td>
+
−
                    <td>1</td>    
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>dH<sub>2</sub>O</td>
+
−
                    <td>14.3</td>
+
−
                    <td>18.0</td>
+
−
                    <td>33.5</td>    
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>Buffer</td>
+
−
                    <td>5</td>
+
−
                    <td>-</td>
+
−
                    <td>10</td>    
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>DNA</td>
+
−
                    <td>touch of colony</td>
+
−
                    <td>touch of colony</td>
+
−
                    <td>touch of colony</td>    
+
−
                </tr>
+
−
            </table>
+
  
 +
<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>PCR Machine Cycling Times and Temperatures: <i>E. coli</i> and <i>Sinorhizobium Plasmids</i></h3>
+
<div class="DatePeriod" id="Week11">
 +
  <h3>Week 11 - August 31- September 4    </h3>
 +
  <ul>
 +
  <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/2015/3/34/Ecppx_KoPPK_digests-XbaI.SpeI_.PNG" height="25%" width="25%"/>
 +
  <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:Tests to see if fluorimetry is a viable means to measure polyphosphate in the cells</li>
 +
  <li>03/09:Slides for fluorescence microscopy prepared using DAPI staining</li>
 +
  <li>04/09:Microscopy reveals polyphosphate chains are visible - success!! </li>
 +
  <li>04/09:Miniprep of ApPst colonies #22, 23, 24, 37, 38, 39. (tested concentraions with nanodrop)</li>
 +
  <li>04/09:Double digest of mini-prepped ApPst with EcoR1 and Pst1</li>
 +
  <li>04/09:Yet another phosphate assay with formic acid done (same strains) - decent results </li>
 +
  </ul>
 +
</div>
  
−
<table>
+
<div class="DatePeriod" id="Week12">
−
    <tr>
+
  <h3>Week 12 - September 7-11  </h3>
−
        <td colspan="2">Step</td>
+
  <ul>
−
        <td>Temperature</td>
+
  <li>07/09:Phosphate assay team analysed water samples from around the world- Israel, Egypt...</li>
−
        <td>Time</td>
+
  <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>
−
    </tr>
+
  <li>08/09:Colony PCR on competent cells transformed with PstC and PPK</li>
−
    <tr>
+
  </ul>
−
        <td colspan="2">Initial Denaturation</td>
+
</div>
−
        <td>95</td>
+
−
        <td>5:00 min</td>
+
−
    </tr>
+
−
    <tr>
+
−
        <td rowspan="3"><br><br>30 Cycles</td>
+
−
        <td>Denaturation</td>
+
−
        <td>95</td>
+
−
        <td>15 sec</td>
+
−
    </tr>
+
−
    <tr>
+
−
        <td>Annealing</td>
+
−
        <td>58</td>
+
−
        <td>15 sec</td>
+
−
    </tr>
+
−
    <tr>
+
−
        <td>Extension</td>
+
−
        <td>72</td>
+
−
        <td>2-4 (1 min per kb)</td>
+
−
    </tr>
+
−
    <tr>
+
−
        <td colspan="2">Final Extension</td>
+
−
        <td>72</td>
+
−
        <td>5-10 min</td>
+
−
    </tr>
+
−
</table>
+
−
 
+
−
Load on 1% agarose gels and run electrophoresis tank until the coloured bands are near the bottom of the gel. Use U:genius machine to photograph and visualise bands.
+
−
</p>
+
  
−
<h2>Digestion Protocols</h2>
+
<div class="DatePeriod" id="Week13">
−
 
+
  <h3>Week 13 - September 13-18  </h3>
−
For each of the following digestions add:
+
  <ul>
−
<ol>
+
  </ul>
−
<li>1 uL Restriction Enzyme</li>
+
−
<li>1 ug DNA</li>
+
−
<li>5 uL 10X NEBuffer</li>
+
−
<li>dH<sub>2</sub>O to make up 50 uL volume</li>
+
−
</ol>
+
−
<p>For the digestions with SpeI, XbaI, PstI and EcoRI-HF, incubation time is 5-15 minutes at 37°C. Inactivation of XbaI and EcoRI-HF takes 20 minuties at 65°C. SpeI and PstI are inactivated at 80°C for 20 minutes. SpeI, XbaI ,and EcoRI-HF have 100% Buffer activity with either NEBuffer 2.1 or Cutsmart while PstI digestions work best with 3.1</p>
+
−
 
+
−
<h2>Mini-Prep or Plasmid Purification</h2>
+
−
<ul>
+
−
<li>Harvest bacterial cells<br>
+
−
1. Pellet 20ml of saturated E. coli for 60 seconds  at 11,000 x g.<br>
+
−
2. Discard supernatant and remove as much liquid as possible.</li>
+
−
<li>Lyse cells<br>
+
−
1. Add 500ml resuspension buffer P1 and resuspend cell pellet by vortexing.<br>
+
−
2. Split the solution into two 1.5ml microcentrifuge tubes.<br>
+
−
3. Add 250μl lysis buffer 2. <br>
+
−
4. Mix gently by inverting tube 8 times. <br>
+
−
5. Incubate at room temperature for five minutes or until lysate appears clear.<br>
+
−
6. Add 300μl neutralization Buffer 3.<br>
+
−
7. Mix thoroughly by inverting tube 8 times.</li>
+
−
<li>Clarification of lysate<br>
+
−
1. Centrifuge for 5 minutes at 11,000 x g at room temperature<br>
+
−
2. Put 500μl of Buffer PW1 per 1.5ml microcentrifuge tube used in heat block heated to 50<sup>օ</sup>C</li>
+
−
<li>Bind DNA<br>
+
−
1. Place ISOLATE II Plasmid Mini Spin Column in a 2ml Collection Tube<br>
+
−
2. Pipette a maximum of 750μl of clarified sample supernatant onto column<br>
+
−
3. Incubate at room temperature for 2 minutes.<br>
+
−
4. Centrifuge for 1 minute at 11,000 x g and discard flow-through.<br>
+
−
5. Repeat stage 4 using the same ISOLATE II Plasmid Mini Spin Column and 2ml Collection Tube with the clarified sample supernatant from the other 1.5ml microcentrifuge tube from the same sample.</li>
+
−
<li>Wash silica membrane<br>
+
−
1. Add 500μl Wash Buffer Pw1<br>
+
−
2. Centrifuge for 1 minute at 11,000 x g <br>
+
−
3. Add 600μl Wash Buffer PW2 (supplemented with ethanol)<br>
+
−
4. Centrifuge for 1 minute at 11,000 x g <br>
+
−
5. Discard flow-through and reuse Collection Tube</li>
+
−
<li>Dry silica membrane<br>
+
−
1. Centrifuge for 2 minutes at 11,000 x g, to remove residual ethanol<br>
+
−
2. Place ISOLATE II Plasmid Mini Spin Column in a 1.5ml microcentrifuge tube.</li>
+
−
<li>Elute DNA<br>
+
−
1. Add 50μl Elution Buffer P directly on the top of the silicon matrix<br>
+
−
2. Incubate at room temperature for 2 minutes<br>
+
−
3. Centrifuge for one minute at 11,000 x g.</li>
+
−
</ul>
+
−
 
+
−
 
+
−
<div id="phosphate assay">
+
−
        <h2 id="phosphate assay"> Phosphate Assay</h2>
+
−
        <h3> Reagent preparation</h3>
+
−
        <ul>
+
−
            <li>
+
−
                <spam style="font-color:orange">Phosphate Reagent:</spam> 15 ml of colorimetric dye. Ready to use as supplied. Equilibrate to room temperature before use. There may be a small amount of precipitate visible which does not affect the assay performance. Store at room temperature. Actually orange.
+
−
 
+
−
            </li>
+
−
            <li>
+
−
                <span style="font-color:orange">Phosphate Standard: </span>500uL of 10mM Phosphate standard. Ready to use as supplied. Equilibrate to room temperature before use. Store at room temperature. Actually Clear.
+
−
            </li>
+
−
        </ul>
+
−
        <h3> Standard preparation</h3>
+
−
        <h4>Materials</h4>
+
−
        <ul>
+
−
            <li>
+
−
                <span style="color:green">Phosphate Standard
+
−
               
+
−
                </li>
+
−
                <li>
+
−
                    <span style="color:blue">Phosphate Reagent
+
−
 
+
−
                    </li>
+
−
                    <li>Distilled Water</li>
+
−
                </ul>
+
−
            </h4>
+
−
            <h4>Procedure</h4>
+
−
            <ol>
+
−
                <li> Dilute 5ul of supplied Phosphate Standard into 495ul of dH2O in a 1.5ml Eppie. Label ‘S’ </li>
+
−
                <li> Make further dilutions to construct the curve. Suggested values are below. Make these in 1.5ml Eppendorfs</li>
+
−
            </ol>
+
−
            <table>
+
−
                <tr>
+
−
                    <td>Volume S/ ul</td>
+
−
                    <td>Volume dH2O/ ul</td>
+
−
                    <td>Concentration/ nMol/200ul(well)</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>0</td>
+
−
                    <td>600</td>
+
−
                    <td>0</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>30</td>
+
−
                    <td>570</td>
+
−
                    <td>1</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>60</td>
+
−
                    <td>540</td>
+
−
                    <td>2</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>90</td>
+
−
                    <td>510</td>
+
−
                    <td>3</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>120</td>
+
−
                    <td>480</td>
+
−
                    <td>4</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>150</td>
+
−
                    <td>450</td>
+
−
                    <td>5</td>
+
−
                </tr>
+
−
            </table>
+
−
            <ol>
+
−
                <li>Pipette 200ul of each curve sample into a well.</li>
+
−
                <li>Add 30ul of Phosphate Reagent to each well</li>
+
−
                <li>Leave to equilibrate for 30 mins at room temperature. N.B the reagent is light sensitive so store in a dark room while equilbrating</li>
+
−
            </ol>
+
−
            <h3>Sample preparation</h3>
+
−
            <p>N.B. Make sure our sample is diluted to ensure the readings are within the standard value range.</p>
+
−
            <p>
+
−
                <u>Steps for cell (adherent or suspension) samples:</u>
+
−
            </p>
+
−
            <ol>
+
−
                <li>Harvest the amount of cells necessary for each assay (initial recommendation = 2 x 10^6 cells).</li>
+
−
                <li>Wash cells with cold TBS (kept in 4oC fridge, stable for 3 months). </li>
+
−
                <li>Resuspend the cell pellet in 150 µL of Assay Buffer (TBS) on ice. </li>
+
−
                <li>Homogenize cells quickly by pipetting up and down a few times. </li>
+
−
                <li>Sonicate cells for 50 seconds 3X at high setup (one cycle = 30 s sonication – 10 s break – 10 s sonication – 10 s break). </li>
+
−
                <li>Centrifuge sample for 15 minutes at 4°C at top speed using a cold microcentrifuge to remove any insoluble material. </li>
+
−
                <li>Collect supernatant and transfer to a clean tube. </li>
+
−
            </ol>
+
−
            <ul>
+
−
                <li>Keep on ice</li>
+
−
            </ul>
+
−
            <p>
+
−
                <u>Tris-Buffered Saline (TBS) Recipe</u>
+
−
            </p>
+
−
            <ol>
+
−
                <li>50 mM Tris-Cl, pH 7.5</li>
+
−
                <li>150 mM NaCl</li>
+
−
                <li>To prepare, dissolve 6.05 g and 8.76 g NaCl in 800 mL of H2O. </li>
+
−
                <li>Adjust pH to 7.5 with 1 M HCl </li>
+
−
                <li>Tris Make volume up to 1 L with H2O.</li>
+
−
                <p>N.B. TBS is stable at 4°C for 3 mo.</p>
+
−
            </ol>
+
−
            <h3>Assay procedure and detection</h3>
+
−
            <p> Equilibrate all materials and prepared reagents to room temperature prior to use. It is recommended to assay all standards, controls and samples in duplicate.</p>
+
−
            <p>Set up Reaction wells: </p>
+
−
            <ol>
+
−
                <li>Standard wells = 200 µL standard dilution. </li>
+
−
                <li>Sample wells = 1 – 100 µL samples (adjust volume to 200 µL/well with ddH2O). </li>
+
−
                <li>Background control sample wells = 1 – 100 samples (adjust volume to 200 µL/well with ddH2O). </li>
+
−
                <li>Add 30 µL of Phosphate Reagent to standard, sample and background control wells. </li>
+
−
                <li>Mix and incubate at room temperature for 30 minutes protected from light. </li>
+
−
                <li>Measure output at OD = 650 nm on a microplate reader.</li>
+
−
            </ol>
+
−
     
+
−
 
+
−
<h3> Glassmilk recipe </h3>
+
−
<ol> <li>Stir 400g silica 325 in 800 ml of dH2O</li>
+
−
<li>Leave to settle for 90 minutes</li>
+
−
<li>Remove supernatant and centrifuge 6000 rpm for 10 minutes</li>
+
−
<li>Resuspend pellet in 250 ml dH2O</li>
+
−
<li>Add conc. Nitric Acid to 50%</li>
+
−
<li>Stir and heat to a boil gently</li>
+
−
<li>Stir to RT gently</li>
+
−
<li>Centrifuge @ 6000 rpm for 10 minutes (make sure you use either glass or polypropylene tubes, polystyrene tubes can’t withstand over 3000g) </li>
+
−
<li>Resuspend in 250 ml dH2O</li>
+
−
<li>Repeat spinning and washing until pH is neutral</li>
+
−
<li>Resuspend pellet to make 50% by volume slurry</li>
+
−
</ol>
+
−
<p>Aliquot and store indefinitely at RT</p>
+
−
+
−
 
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<h2>Growth Assay - Plate Reader</h2>
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<h4>Materials</h4>
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<ul>
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<li>96 well plate</li>
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<li>10x MOPS Media</li>
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<ul><li>dilute to 1X</li></ul>
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<li>K<sub>2</sub>HPO<sub>4</sub> (dipotassium phosphate)</li>
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<ul><li>@0.1 mM for low P</li>
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    <li>@1.32 mM for high P - suggested by MOPS media recipe</li>
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    <li>Molar mass- 174.2 g/mol </li>
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</ul>
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<li>4 mg/mL glucose</li>
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<li>strains of bacteria</li>
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</ul>
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<h4>Purpose</h4>
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<p>-used to measure bacterial density by tracking absorbance at intervals of 30 minutes for 48 hours (220 rpm, 37°C) </p>
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<h4>Procedure</h4>
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<p>Day 1</p>
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<ol>
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<li>prepare the phosophate concentrations and filter sterilize them</li>
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<li>prepare the glucose solution and filter sterilize them</li>
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<li>prepare 1X MOPS (from 10X solution) filter sterilize them </li>
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<li>prepare overnights</li>
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<ol><li>5ml of LB in a BD falcon tube</li>
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<li>take a colony off the plate with a sterile toothpick and put in falcon tube </li>
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<li>leave overnight at 37°C in a shaker </li></ol></ol>
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<p>Day 2</p>
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<li>transfer overnight culture into 1.5 mL eppendorf tubes</li>
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<li>spin down overnights - 3 min at 4K </li>
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<li>wash out with MOPS and resuspend with 1 mL MOPS</li>
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<li>repeat spin step and resuspend again </li>
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<li>measure OD<sub>650</sub> of a 10 fold dilution of cells with a spectrophotometer blanked with MOPS </li>
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<li>calculate the amount of inocula required  for a final OD<sub>650</sub> of 0.4 in 500 uL</li>
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<li>set up wells - 2 uL phosphate, 5 uL cells, 193 uL MOPS </li>
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Revision as of 14:37, 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