Team:Oxford/Notebook

iGEM Oxford 2016 - Cure for Copper

Notebook

Introduction

This page documents all the experiments we carried out in the wet lab as a part of our project. The details of the process we carried out can be found on our protocols page, and the chemicals we used can be found on the chemicals page. The interlab project was carried out alongside the rest of the wet lab but we have recorded it separately.

Week 1

Day 1

Preparation of Stock Solutions

gBlocks

3 of our gBlocks from IDT had arrived: pCopA MymT HH (pc), MymT sfGFP HH (mg) and TAT Csp1 HH (tc). These arrived in the form of a solid, white powder. We resuspended the gBlocks in different volumes of MilliQ, depending on the weight delivered, to give a stock solution of 10 ng/l.

Primers. The forward and reverse primers from IDT came as 32.4 nmol and 23.8 nmol of solid respectively. Forward: 5’-CGACTTGATCACGTAGAATTC-3’. Reverse: 5’-ACACGATCGATATAACTGCAGC-3’. For our stock solutions, the primers were suspended in different volumes of MilliQ to give 100M.

Preparation of reaction solutions. gBlocks: Following resuspension, 10l of each DNA solution was added to 90l MilliQ (1:10 dilution) in an Eppendorf to give a final concentration of 1 ng/l and final solution volume of 100l.

Primers: Following resuspension, 10l of each primer solution was added to 90l MilliQ (1:10 dilution) in an Eppendorf to give a final concentration of 10M and final solution volume of 100l.

Day 2

Polymerase Chain Reaction (PCR). 25l reactions were run according to the NEB Q5 High-Fidelity 2X Master Mix PCR protocol. * 98OC denaturation temperature used due to the high stability of the Q5 polymerase. ** 60OC annealing temperature used on Chris’s recommendation. *** 30s elongation time used as maximum DNA length was 936bp and a PCR takes approximately 20-30s to extend a DNA sequence by 1000bp.

Gel Electrophoresis of PCR-Amplified gBlocks

A 1% agarose gel was prepared according to the agarose gel preparation protocol. A DNA ladder mix was prepared in an eppendorf. The 25l solutions of PCR-Amplifed gBlocks were mixed with 5l loading dye in order to visualize their migration. 20l of this solution was then loaded into each gel well. The gel was run with a potential difference of 100V applied across the electrodes for approx. 40 minutes. Following separation, the gel was transferred to a bath of EtBr for staining. It was left shaking for 20 minutes. Success! The bands produced corresponded to the expected DNA sizes. These were excised using a razor blade and placed in labelled eppendorfs for freezing overnight.

Day 3

Gel extraction of PCR-Amplified gBlocks from agarose gel

The extraction was performed using the Qiagen QIAquick Gel Extraction Kit protocol. 540l of Buffer QG was added to each tube in 2. In 9, 30l MilliQ was used rather than 50l buffer EB on Chris’s recommendation. The tubes were spun with lids open, hence, they were spun with the lids positioned such that would not flip around during centrifugation, away from the direction of spinning.

Preparation of MymT HH Stock Solution. Another gBlock, MymT HH, arrived from IDT and was resuspended as described in Week 1: Day 1. 424ng was delivered, so 42.4l MilliQ was added to give 10ng/l.

Preparation of MymT HH reaction solution. Same procedure as Week 1: Day 1.

PCR of MymT. Same program as Week 1: Day 2 used, apart from using a 62OC annealing temperature. gBlocks from yesterday also run to see whether the higher annealing temperature eliminated the smears observed in the PCR from Day 2.

MymT sfGFP HH and TAT Csp1 ran slightly better under the new conditions. No result from pCopA MymT HH. No template controls run adjacent under same name as DNA produced no result, as expected. MymT HH produced a large smear. Will run again in future.

Day 4

PCR of MymT

Day 5

Open day, no wet lab.

Week 2

Day 1

PCR of MymT. After no success last week, a new working reaction solution was made up in case the previous reaction solution had been contaminated. Run under new conditions but still a smear over most molecular weights. Carry out again.

PCR, gel extraction and restriction enzyme digest of shipping vector. Using shipping vector from the team last year for our plasmids. Ran a PCR using standard conditions to amplify amount of the vector. Gel Extraction of plasmid using the Qiagen QIAquick Gel Extraction Kit protocol. Restriction enzyme digest of extraction using same protocol as followed in Week 1: Day 4. 10l of plasmid used instead of 25l.

Gel extraction of restriction digest incubations. Upon completion of the restriction digest incubation, the gBlocks and the plasmid backbones were purified using the Qiagen QIAquick Gel Extraction Kit protocol. We quantified the amount of DNA using NanoDrop 1ul of water and tissue were initially used to clean the NanoDrop stage. A blank reading was made using 1ul of MilliQ. 1ul of each DNA sample was measured for concentration. The DNA was ligated with the shipping vector and left overnight at 16OC.

Day 2

Antibiotic stock made. Shipping plasmid contains gene conferring resistance to chloramphenicol, so chloramphenicol antibiotic stock made up. Reaction mixture composed of 300mg chloramphenicol, 0.5ml MilliQ, and 9.5ml ethanol (prevents freezing). After making solution, filter pipette tips used to produce 20 x 0.5ml aliquots of chloramphenicol solution for later use.

Set up agar plates. LB agar melted to produce a pourable liquid. After melting, chloramphenicol from stock solution added at 1:1000 dilution. Plating in flow cupboard. Plates left to set for 30 minutes.

Transformation of E. coli cells with ligated plasmid DNA. Competent E. coli cells removed from freezer at -80OC and thawed on ice. Transformation protocol followed. 2 plates for each DNA sample (MymT sfGFP HH, TAT Csp1 HH, pCopA MymT HH sample 1, and pCopA MymT HH sample 2). First plate produced by spreading 100l of the cell solution onto one plate. Second plate produced after spinning down the remaining solution and resuspending in LB broth and plating, resulting in a more concentrated plate. Plates left to incubate overnight at 37OC.

Miniprep of CusC. Minipreparation: isolation of plasmid DNA from bacteria. Completed using the QIAprep Spin Miniprep Kit protocol. Resulting solution placed in freezer at -20OC

Day 3

Growth and Culture of bacteria. Plates incubated overnight all contain colonies. More are present on the concentrated plates. 2 colonies from each plate are selected (not too large or too small) and incubated in test tubes with 5ml LB broth and chloramphenicol in 37OC shaker overnight. Aim of the procedure is to increase the number of plasmids containing our biobricks.

PCR of MymT HH was unsuccessful again.

Preparation of pCopA TAT Csp1 sfGFP HH (ptcg) Stock Solution. Another gBlock, pCopA TAT Csp1 sfGFP HH, arrived from IDT and was resuspended as described in Week 1: Day 1. 1000ng was delivered, so 100l MilliQ was added to give 10ng/l.

Preparation of MymT HH reaction solution - Same procedure as Week 1: Day 1.

PCR of pCopA TAT Csp1 sfGFP HH - Unsuccessful – producing a large DNA smear over many different DNA sizes.

Day 4

PCRs of pCopA TAT Csp1 sfGFP HH and MymT HH - All unsuccessful.

Miniprep of pCopA MymT HH, MyMT sfGFP HH and TAT Csp1 HH completed using the QIAprep Spin Miniprep Kit protocol. Resulting solution placed in freezer at -20OC. Diagnostic gel of pCopA MymT HH, MyMT sfGFP HH and TAT Csp1 HH. Aim was to check that our biobrick inserts had been successfully incorporated into the plasmid, by digesting with the restriction enzyme. Gel set up.

Day 5

PCRs of pCopA TAT Csp1 sfGFP HH and MymT HH - All unsuccessful.

Week 3

Day 1

Only interlab wet lab,

Day 2

Only interlab wet lab.

Day 3

PCRs of pCopA TAT Csp1 sfGFP HH and MymT HH - All unsuccessful. Negative control gave spear – possible risk of contamination Transformed parts: DspB, DspBx, DsbA-DspB, and DsbA-DspBx to send to XBU China.

Day 4

PCRs of TAT Csp1 HH (tc) and negative control to check no contamination (positive control and negative control). Successful! No DNA in negative control, bands where expected to be in positive control. Picked colonies from DspB, DspBx, DsbA-DspB, and DsbA-DspBx.

Day 5

Mini prepped sequences for XBU China (DspB, DspBx, DsbA-DspB and DsbA-DspBx). Isolated 2 colonies per part (just for back-up). Completed using the QIAprep Spin Miniprep Kit protocol. Resulting solution placed in freezer at -20OC. Sent parts off to China

Sent sequences off for sequencing: CSP1A – VF2, CSP1A – VR, CSP1B – VF2, CSP1B – VR, MGA – VF2, MGA – VR, MGB – VF2, MGB – VR, PMA – VF2, PMA – VR, PMB – VF2, PMB – VR, pCusC – CC forward, pCusC – CC reverse. Required concentrations: Plasmid DNA - 5l of 100ng/l, Primers - 5l of 3.2pmol/l. All failed except the pCusCs.

Week 4

Day 1

pCopA MymT sfGFP HH (pmg) nad TAT Csp1 sfGFP HH (cg) arrive. Resuspend (both arrived as 1000ng sample), dilute to 10ng/l in tube then take dilute by 1 in 10 to get a 1ng/ml stock then add 1l of this to the PCR mixture.

PCR pmg and cg using standaed concentrations and volumes. 25 cycles, 95C for 15s, 62C for 15s, 72C for 1 min. Results: both produced smears (NTC = no result)

Day 2

PCR pmg and cg. 64C, 1 min elongation time and 66C, 1 min elongation time. pmg64, pmg66 and cg64 all produced smears, cg66 showed nothing

PCR ptcg because new primers arrived. Ran at 60C with new primers. Success! Gel extract ptcg. Gel volume = 50l

PCR pmg (using new ptcg primers) and cg (using new ptcg reverse primer and usual forward primer). Run at 60C and 62C. All successful!

Day 3

Gel extract cg and pmg, gel volume = 40l

PCR MymT using normal forward primer and new ptcg reverse primer. 60C and 62C, 1 min elongation time. m60 = smear, m62 = smear but band at 254bp

PCR MymT again at 63C and 68C with 15s elongation time. Both still produced a smear but IDT said this part’s mass spec was messy, band is present

Gel extract MymT HH, gel volume = 50l

PCR ptcg and pmg using combination of old and new primers: Ptcg1 = old forward, new reverse, Ptcg2 = new forward, old reverse, Pmg1 = old forward, new reverse, Pmg2 = new forward, old reverse. 2s gave better results (used 2s for gel extraction and ligation)

Day 4

Ligation of cg (new forward and old reverse primers) and MymT (m) (new forward and old reverse primers), cut with EcoRI and SpeI and used CutSmart buffer.

Gel extract ptcg and pmg, gel volume = 50l

Day 5

Digest and ligate ptcg2 and pmg2 (both using new forward and old reverse) using PstI and XbaI. Diagnostic gel for cg and Mym using EcoRI and SpeI

Getting things into pBad from the shipping plasmid. PCR amplify with specific primer pairs from plasmid. 4 reactions using 5 primers.

1. Csp1 into pBad, then cute this into BspHI and Pst1, cut pBad with NcoI and PstI Ex P TAT Csp1 Forward – Tm 64C Ex P Rev – Tm 67C

2. MymT sfGFP into pBad Ex P MymT Forw – Tm 64C Ex P Rev – Tm 67C

3. MymT sfGFP into MymT in pBAD Ex P MymT Forw – Tm 64C MymT only cut Rev – Tm 63C

4. MymT sfGFP into MymT in shipping vector – cut with EcoRI and PstI, cut shipping vector with EcoRI/ and PstI MymT shipping forward – Tm 61C MymT only cut – Tm 63C

PCR conditions, 95C 02:00, Then 25x {95C 00:15, 62C 00:15, 72C 00:30} Then 72C 01:00 and hold at 10C. Results: Ran off gel but then re-ran

Week 5

Day 1

pg arrived, band size is 1391 bp. Resuspended.

PCR pg. pg1 – old forward, new reverse primers, pg2 – new forward, old reverse primers. Both at 60C, 1 min elongation. Ran gel of pg PCR products. pg1 – successful, cut from gel but pg2 – unsuccessful, multiple bands produced

Digest and ligate Csp1 into pBad. Cut parts with BspHI and PstI and cut plasmid (pBAD) with NcoI and PstI using CutSmart buffer. Digest and ligate MymT sfGFP ino pBad. Cut parts with BspHI and PstI and cut plasmid (pBAD) with NcoI and PstI using CutSmart buffer. Digest and ligate MymT sfGFP into MymT in pBad. Cut parts with BspHI and PstI and cut plasmid (pBAD) with NcoI and PstI using CutSmart buffer.

Day 2

Diagnostic gel for cg (16) and m (16)

Gel extract pg, gel weight = 10mg. Enzyme digestion of pg gel extract using EcoRI and SpeI and CutSmart buffer. Ligation of pg into shipping plasmid.

Day 3

PCR mg from sequenced plasmid (“3” and “4”). To get m out cut 3 with BspHI and PstI and cut 4 with EcoRI and PstI. Conditions = 95C, 62C annealing, 25x cycles

Digest ptcg and pmg using EcoRI and SpeI. Gel extract 3 and 4, Gel volume = 40mg (used 2% gel)

Miniprep pmg (14) and ptcg (14), ran diagnostic gel on miniprep product. Unsuccessful.

Transformations of pg using chloroamphenicol and of tc and mg (into pBAD) using amp.

Day 4

Digest ptcg (1 and 2) and pmg (1 and 2), and 3 and 4. Digest shipping plasmid with M3 and M4 inside it. M3 with BspHI and PstI, M4 with EcoRI and PstI.

Re-digestion of unsuccessful transformation minipreps. Ptcg1: old forward primer, new reverse primer, use EcoRI and SpeI and CutSmart buffer. Ptcg2: new forward primer, old reverse primer, use PstI and XbaI and CutSmart buffer. Pmg1: old forward primer, new reverse primer, use EcoRI and SpeI and CutSmart buffer. Pmg2: new forward primer, old reverse primer use PstI and XbaI and CutSmart buffer. M3 into pBAD use BspHI and PstI and CutSmart buffer. M4 into shipping plasmid use EcoRI and PstI and CutSmart buffer

Colonies picked from tc, mg (amp – pBAD) and pg (chloroamphenicol – shipping).

Day 5

Digest shipping plasmid to use for ligation of M3 and M4. Digest one plasmid with BspI and PstI. Digest 2nd with EcoRI and PstI. Run gel of these digested shipping plasmids.

Ligation. Pmg1 – EcoRI and SpeI, Pmg2 – PstI and XbaI, Ptcg1 – EcoRI and SpeI, Ptcg2 – PstI and XbaI, M3 (pBAD) – BspHI and PstI, M4 (shipping plasmid) – EcoRI and PstI.

Miniprep tc, mg, cg and pg, run diagnostic digest and gel. tc and mg in pBAD – using BamHI and PstI. cg and pg in shipping plasmid – using EcoRI and SpeI

Day 7

Picked Mg1655 colonies.

Week 6

Day 1

Transform ptcg and pmg 2 plates each, therefore 4 plates

Feedback pCusC mKATE (fck) arrived 1000ng delivered → add 100ul to give 10ng/ul After resuspension, dilute 1 in 10 to give 1ng/ul

PCR fck fck,on 60 = old forward, old reverse primers, 60p annealing fck,on 62 = old forward, old reverse primers, 62o annealing fck,on 60 = old forward, new reverse primers, 60o annealing fck, on 62 = old forward, new reverse primers, 62o annealing Results: fck on 60 and fck on 62 worked

Gel extraction for fck

Day 2

Picked colonies from pmg and ptcg

Day 3

Miniprep ptcg, pmg and pm

Diagnostic gel for ptcg and pmg Using PstI and EcoRI Expected band sizes = 1824 and 1565, respectively

Sent ptcg and pmg for sequencing

Day 4

Sequencing results ptcg → didn’t work pmg → didn’t work cg (in shipping plasmid) → didn’t work tc → didn’t work pg → has promoter, no mismatches, truncated mg → good

Next steps: Re-transform fck, PCR ptcg, pmg, cg and tc, Pick M3 colonies, Transform pg and mg into MG1655

Old shipping plasmid possibly contaminated, digest new. Using part from last year (Art-175). Component: Art-175 5ul, 10xbuffer 5ul, EcoRI 1ul, SpeI 1ul and MilliQ 38ul. Set up digest at 37o for 2 hours

Next steps: Heat inactivate at 80o for 10 mins. Dephosphorylate with 1ul antarctic phosphatase 30 mins at 37o. Run gel. Excise 2kb band.

PCR tc, cg, ptcg and pmg. Tc – 60o annealing, old forward, old reverse. Cg – 62o annealing, old forward, new ptcg reverse. Ptcg – 60o annealing, new forward, old reverse. Pmg – 60o, new forward, old reverse.

Gel for tc, cg, ptcg and pmg Expected band size = 545, 1277, 1824 and 1565 bp, respectively Results: tc = very smeary, other 3 to be extracted

Day 5

PCR ptcg and pmg. ptcg = new forward, old reverse primers,60o annealing temperature

Transform pg and mg into MG1655, pg uses chloroamphenicol, mg uses ampicillin.

Week 7

Day 1

Measuring fluorescence in 96 well plate of pg

Transformation of mg(pBAD) into MG1655 using ampicillin.

Pick 4x colonies from pg plate using chloroamphenicol.

Pick 4x colonies from MG1655, no antibiotic.

Ligate ptcg, pmg, tc, cg and fck

Day 2

Miniprep M3 using ampicillin.

Digest and diagnostic gel using EcoRI and SpeI. Expected band size = 254bp Came back incorrect

Pick colonies for pCusC, MG1655 negative control, MG into pBAD using chloroamphenicol, no antibiotic and amphicillin, respectively. 4 colonies each

Transformations of fck, ptcg, pmg, tc and cg into DH5-alpha, chloroamphenicol need 10 plates. Not successful.

Day 3

Transformations of fck, ptcg, pmg, tc and cg into DH5-alpha. Results: unsuccessful for all except cg. Chloroamphenicol

Transformation of mg(pBAD) into MG1655, Ampicillin, Successful.

Digestion and diagnostic gel of M3 (1-6). On gel, expected band size = 287bp. Used 2% agrose gel.

PCR m out of mg shipping vector. Expected band size = 196bp Successful Extracted

Day 4

Miniprep cg

Diagnostic gel for cg, use EcoRI and SpeI. Expected band size = 1250bp

Plate reader experiment for pg and negative control, followed protocol. 50ul LB (not 40ul) OD 600

Transforms: Pmg – chlorophenicol into DH5alpha, Fck – chloroamphenicol into DH5alpha, Ptcg – chloramphenicol into DH5alpha, mEx – ampicillin into DH5alpha, pcg – chloramphenicol into DH5alpha, pBAD vector no insert = ampicillin MG1655.

Day 5

Miniprep cg . Diagnostic gel for cg. Digest using EcoRI and SpeI. Expected band size = 1250bp

Plate reader experiment for pg and negative control. Followed protocol. 50ul LB (not 40ul). OD 600

Transforms: Pmg – chlorophenicol into DH5alpha. Fck – chloroamphenicol into DH5alpha. Ptcg – chloroamphenicol into DH5alpha. mEx – amphicillin into DH5alpha. pcg – chloroamphenicol into DH5alpha. pBAD vector no insert = amphicillin MG1655

Week 8

Day 1

PCR ptcg, pmg, cg and fck. For ptcg – use old forward (EcoRI), new reverse (SpeI), 60o annealing. For pmg – use old forward (EcoRI), new reverse (SpeI), 60o annealing. For cg – use old forward (EcoRI), new reverse (SpeI), 60o annealing. For fck – use old forward (EcoRI), new reverse (SpeI), 60o annealing. All 1 min elongation time and ran 2 of each. Results: ptcg, pmg, cg and fck successful

Gel extract all 4 parts and mymT in expression (mEx)

Digestion for all 4 parts and mEx using EcoRI and SpeI for ptcg, pmg, cg and fck, XbaI and PstI for mEx

Ligation of ptcg, pmg, tc, cg, pcg, mEx and fck

Nanodrop cg and fck, 7.6 and 18.8, respectively. Start from PCR stage.

PCR m out of mg (retry) 62o annealing – 15s, 72o elongation – 15s

Day 2

Started using improved cloning procedures to attempt to get everything working

Starting from PCR with tc, cg, ptcg, pmg, pcg and fck. Old forward, new reverse primers for cg, ptcg, pmg, pcg and fck. Old forward, old reverse primer for tc.

Transform Fla-Art175 (part from last year) and tc(shipping) into DH5-alpha, use chloroamphenicol. Fla-Art175 = 1120 bp. tc = 545 bp. PSB1C3C shipping plasmid backbone = 2070bp

Transform pmg, Mex and tc (pBAD) into DH5-alpha, use chloroamphenicol for pmg and use ampicillin for mEx and tc.

Pick colonies of pBAD (no insert) to make pBAD stock. Pick x8

Day 3

Miniprep pBAD inset x6. Ran x2 samples from each picked colony. Digest with NcoI and PstI. Only digested using sample from 1a.

PCR tc, cg, ptcg, pcg 1, fcg 2. tc - use old forward, old reverse, 62o annealing. cg - use old forward, new reverse, 60o annealing. ptcg - use old forward, new reverse, 62o annealing. pcg 1 - use old forward, new reverse, 60o annealing. fcg 2 - use old forward, new reverse, 62o annealing.

Gel extract to make pBAD stock solution. Used 1a colony. Send undigested plasmid for sequencing. Put 1a gel in freezer. Gel extract if sequencing comes back correct.

Pick colonies of: Fla-Art175 (x4, chloroamphenicol), Tc (shipping) for vector (x4, chloroamphenicol), mEx (x6, amphicillin), Tc in pBAD (x6, amphicillin).

PCR tc out of shipping tc2 using ExP Rev and Exp Tat Csp1 primers, 62o annealing. Ran gel → got one band on each so can do PCR purification rather than gel extraction

Day 4

Miniprep tc for shipping, Fla-Art175 shipping and mEx. Tc shipping = 1a, 1b, 2a, 2c, Fla-Art175 shipping = 3a, 3b, 4a, 4c, mEx = A, B, C, D, E

Digestion for above parts, 1a to 4b using EcoRI and SpeI. A to E using BamHI and PstI (use 2% gel)

Gel for Mex (A to E) came back successful, nanodropped A to E. C had highest value (35.5) → sent for sequencing

Gel for 1a-4a, 1a = tc, 2a = tc, 3a = Fla-Art175, 4a = Fla-art175. Kept 1a and 2a. Set up more to digest overnight

Pick colonies for mEx and pg (MG1655)

Day 5

Miniprep mEx (1-4), digest, diagnostic gel, sequence. Nandrop results: 1 = 95.8, 2 = 99.4,3 = 89.3, 4 = 93.5 Discarded 3.

Re-transform pg into MG1655

Run mEx gel, Ran mEx 1-4. Sending mEx2 for sequencing. Keep mEx1 just in case.

Day 7

Made more chloramphenicol

Pick colonies of fcg, cg, ptcg, fck, pcg, pmg and mEx, 6x of each, apart from pmg (only 1x). Chloramphenicol for all except mEx (ampicillin)

Week 9

Day 1

Miniprep cg, fcg and ptcg

Digest cg, fcg and ptcg for diagnostic gel

Transform tc and pmg

Day 2

Pick colonies of mg and pmg, 2x of each, Chloramphenicol

Pick colonies of tc, 1x of each Chloramphenicol

Day 3

Miniprep 2x pmg and 1x tc Results: tc = 75.8, pmg a = 104.0, pmg b = 332.9

Digest tc and pmg a, using EcoRI and PstI. Ran gel with just pmg a One band at around 2000, probably shipping vector so hasn’t worked

Day 4

Send tc2 for sequencing Successful!

Digest pmg a and b from Day 3 using EcoRI and PstI. Expected band size = 1533

Nanodrop pmg a and pmg b Results: 104.0 and 332.9, respectively From gel, pmg b worked so sent off for sequencing

Digest fck 1-6 for diagnostic gel using EcoRI and PstI, both on 1% gel

PCR purify fck 1-6 1 volume 20ul, 3 volume 60ul 60ul QG 20ul isopropanol From gel – fck didn’t work

Day 5

Digest and diagnostic gel of mEx (Freezer has 1,3,4,5 and 6) using BamHI and PstI. Run diagnostic gel – expected size = 264bp

Week 10

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Day 3

Day 4

Day 5

Week 11

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Day 5

Week 12

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Day 5

Week 13

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Day 4

Day 5

Week 14

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Week 15

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Week 16

Day 1

Day 2

Day 3

Day 4

Day 5

Interlab

Day 1

Transformed the 5 interlab parts into DH5a as per the transformation protocol.

Day 2

Transformations mostly unsuccessful. Started making new competent cells.

Day 3

Second day of competent cell preparation, cells frozen and put in the freezer.

Day 4

LB media preparation and re-transformation of the interlab parts.

Day 5

Had colonies from all interlab parts except PC. Picked colonies from all successful plates. Retransformed PC.

Day 6

PC didn't transform again. After measuring the sample we were sent with nanodrop we found the sample has no DNA in it. Requested a second sample from iGEM HQ Nevertheless we did a third transformation for PC. NC and TD1-3 were miniprepped and sent for sequencing.

Day 7

TD1 sequencing successful, others had the wrong part in so retransformed these. PC colonies successful despite nanodrop so picked colonies for these.

Day 8

PC miniprepped, digested and sent for sequencing. Colonies picked for re-transformed TD2, TD3 and NC.

Day 9

PC sequencing unsuccessful. TD2, TD3 and NC miniprepped and sent for sequencing.

Day 10

TD2 sequencing successful, others not. PC, NC and TD3 transformed again

Day 11

PC, NC and TD3 colonies picked.

Day 12

PC, NC and TD3 miniprepped and sent for sequencing.

Day 13

NC and TD3 sequencing successful but PC wrong so retransformed again.

Day 14

Picked colonies for PC.

Day 15

PC was miniprepped and digested. Gel showed it contained the wrong part so we transformed the part from the distribution kit instead.

Day 16

Colonies picked for PC.

Day 17

PC miniprepped, digested and sent for sequencing. Produced more competent cells.

Day 18

PC sequencing correct! All 5 interlab parts were transformed into MG1655

Day 19

Made the calibration curves for the OD600 and the FITC standard curve Colonies picked for all the interlab parts.

Day 20

Tested iGEM's protocol for the OD600 and the fluorescence. Picked more colonies for all 5 parts.

Day 21

Repeated iGEM's protocol and started Oxford iGEM's protocol.

Day 22

Finished Oxford iGEM's protocol and submitted the data obtained using iGEM's method to iGEM