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: Phage contamination scare - total clean of the lab
• 27/07: More BW25113 competent cells made
• 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
• 05/08:Transformation of gibson from 30/07
• 05/08:Q5 Colony PCR of SmPstSCAB, EcPstSCAB, EcPhoESCAB
• 05/08:PCR pf pBC9, pBC29
• 06/08:Mini-prep of pKDM12 and KoPPK, nanodrop
• 06/08: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:Mini-prep of overnight cultures of ApPst, ApPPK (SK-12, BA-91, UW-1)
• 07/08:Used Nanodrop machine to measure concentration of miniprep products
• 07/08:Performed an analytical digest of purified plasmids [ApPst, ApPPK (SK-12, BA-91, UW-1)] 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

• 23/08:Inoculate liquid cultures of EcPPX, EcPPK, pKDM12, KoPPK
• 23/08:Dephosphorylate lacZ #1,2,3 with Antarctic Phosphatase
• 24/08:Run double digest of 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:Transformed BW25113 lacZ #1 cells with EcPPK, EcPPx, KoPPK, pKDM12 and plated
• 27/08:Made grid plates of EcPhoE, EcPst and ApPst to screen colonies that have been transformed with the respective plasmid

• 28/08: 3 in 1 GoTaq Colony PCR of EcPhoE, EcPst and ApPst (use 3 colony samples per PCR tube to save reagents and reduce the number of samples to be run)

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