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<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: Article 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: Article 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>Article 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>  

<h3>Week 1 - June 23-27 </h3>

<ul>

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

</ul>

<h3>Week 2 - June 29- July 3 </h3>

<ul>

<li>01/07:Competent cells made</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>03/07:Competent cells were tested with iGEM transformation efficiency kit</li>

</ul>

<h3>Week 3 - July 6-10 </h3>

<ul>

<li>07/07: More agar plates made, next set of competent cell procedure started</li>

<li>07/07: YUfund page done and submitted</li>

</ul>

<h3>Week 4 - July 13-17  </h3>

<ul>

<li>14/07: Vistited Badger Hill Primary School and held a bacteria workshop for the students</li>

<li>15/07: first day of demonstrations of cell transformations and aseptic technique to sixth formers</li>

<li>16/07: second day of demonstrations to sixth formers:analysing transformations and building bioreactors</li>

<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>

<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>

<h3>Week 6 - July 27-31 </h3>

<ul>

<li>27/08:Preparations for glassmilk procedure</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>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>30/07:After several repeats the phosphate assay team managed to get significant data</li>

<li>30/07:Phosphate assay on Keasling strains: pBC9, pBC29 and pKDM12.</li>

</ul>

<h3>Week 7 - August 3-7 </h3>

<ul>

<li>03/08:Plasmid (pBC 29 Keasling collection )loss experiment initiated</li>

<li>05/08:Q5 PCR of SmPst, EcPst, EcPhoE</li>

<li>17/07:Third attempt at growth assay - did not test any Pst or PhoE genes, results ok</li>

<li>06/08:Inoculated overnight cultures of ApPst, ApPPK (SK-12, BA-91, UW-1)</li>

<li>06/08:Ran GoTaq Colony PCR of same colonies of ApPst, ApPPK (SK-12, BA-91, UW-1)</li>

<li>06/08:Ran gel electrophoresis of colony PCR</li>

<li>06/08:Inoculated overnight cultures of BW25113, pkDM12, KoPPK</li>

<li>17/07: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>

<h3>Week 8 - August 10-14    </h3>

<ul>

<li>10/08:Transform competent cells with plasmid (?)</li>

<li>10/08:Ran GoTaq colony PCR of ApPst, screened for correct product, ran gel</li>  

<li>10/08:Performed a gibson assembly of EcPhoE Q5 PCR product (from 05/08)</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>10/08:Ran an overhang-adding step-up PCR of KoPPK, pkDM12, pBC9, pBC29</li>

<li>11/08:Ran a <em>Pseudomonas aeruginosa</em> colony PCR to amplify PaOprO and PaOprP</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>11/08:Glassmilk was made</li>

<li>11/08:NMR test ran on BW25113</li>

<li>12/08:Another phosphate assay run- using more formic acid, not neutralised this time. </li>

<li>12/08:Digested lacZ pSB1C3 with EcoR1, ran gel, and performed gel extraction and purification protocols</li>

<li>12/08:Fifth attempt at growth assay - new plate design with increased phosphate levels</li>

<li>13/08:Digest pAdapt with Sma1</li>

<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>

<h3>Week 9 - August 17-21  </h3>

<ul>

<li>17/08:Make liquid cultures of colonies that were verified with the gel on the 14th(EcPPX, EcPPK, pKDM12) </li>

<li>17/08:Attempt colony PCR of different colonies for gibsons that did not work (EcPst, KoPPK, SmPst, EcPhoE)</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>17/08:Ran gel of PaOprO/PaOprP PCR attempt #1 - no positive results, even control failed</li>

<li>17/08:Glassmilk was used to isolate PolyP during yet another phosphate assay - this also did not work.</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>19/08:Growth median assay on all of KEIO collection for phosphate assay purposes</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>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>20/08:<em>Pseudomonas aeruginosa</em> PCR attempt #3 - changed extension time from 30 to 40 seconds. No positive results. Control failed.</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>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>

<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>

</ul>

<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>

<h3>Week 12 - September 7-11   </h3>

<ul>

<li>07/09:Phosphate assay team analysed water samples from around the world- Israel, Egypt...</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>

<li>08/09:Colony PCR on competent cells transformed with PstC and PPK</li>

</ul>

<h3>Week 13 - September 13-18  </h3>

<ul>

</ul>

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