The following is a weekly description of the experiments carried out each week. To see the protocols click here
(Click to enlarge images)
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
- 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.
- Articlepublished on Mind the Horizon about University of York iGEM
- Lab safety induction was carried out
- 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
- 23-27/07:Dry lab research continues
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
- 07/08: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
- 10/09:Digested pAdapt with SmaI, dephosphorylated with Antarctic Phosphatase, ran gel electrophoresis
- 10/09:Ran Q5 Colony PCR of BW25113 cells with EcPst Primers - bulk up
- 11/09:Prepared BW25113 and KoPPK cells for NMR
Week 13 - September 13-18
- 14/09:Shipped first plate of biobricks to iGEM HQ!
- 14/09:NMR of KoPPK strain
- 14/09:Set up 7 hour growth assay
- 15/09:Set up 6 hour combined growth and phosphate assay
- 15/09:Mini-prep, nanodrop, digestion with EcoR1 and Pst1 of ApPst colonies - no success, realised we needed to let other colonies grow more as Accumulibacter grows slowly
- 16/09:Repeat previous day's protocols with new overnight culture of ApPst
- 16/09:Sonicated cells from the previous day and analysed with a plate reader alongside a media assay.
- 17/09:Shipped second plate of biobricks to iGEM HQ!
- 17/09:Finalised business plan
- 17/09:Sonication of cells and phosphate assay to characterise cells
- 18/09:Final sonication of cells and phosphate assay data analysis
- 18/09:Lab clean-up!
- 18/09:Finished wiki!