- Began toxicity study for first substrates of interest (toluene, xylenes mix)
- Made stock of BL21 DE3
- Made fresh chloramphenicol (Cm) and kanamycin (Kan)
- Prepped chemically competent BL21 DE3
May 19, 2016
- Tested chemi-comp cells with 2016 Distribution Kit chemi-comp plasmids
- Experiment 1 completed at 24 hours growth
May 20, 2016
- 50pg/uL transformation with chemi-comp BL21 DE3 from 5/18 was successful
- 1 colony observed with 20pg/uL transformation
June 7, 2016
- Synthesized DNA from IDT arrived (Synth1, 2, 3, 5, and 6) as well as primer
June 8, 2016
- Amplified pSB1K3 and pSB1C3 from linearized backbone
- Primers: PSB1X3.F and PSB1X3.R (63 °C annealing temp)
- Q5 polymerase
- 25 ng DNA template
- Extension time: 1:30 min
- Expected ~2100 bp for both
- Gel picture
- PCR repeated due to streaks in gel (same conditions)
- Gel picture
- Digested pieces Synth 3, 5, and 6 with EcoRI and XbaI
- 100 ng template for each piece
June 10, 2016
- Amplification of linear backbone from 6/8/16 successful, but pieces extracted from gel were not clean enough to use
- Getting a fresh pSB1C3 BB from registry to amplify from (part BBa_K145014, Plate 4, well 1E)
- Transformed BBa_K145014 into previously prepped BL21 DE3
June 11, 2016
- Troubles with digestion/ligation and Gibson mastermix in house; troubleshooting in progress
June 22, 2016
- Gibson of pSynth1-2, pSynth3, pSynth5, and pSynth6 and transformation into Top10 BL21 DE3 successful
- Temperature optimized all primers when doing PCR confirmations of pSynthX + BL21 DE3
- Q5 polymerase
- 1 colony DNA template
- Extension time: 2:00 min
- Expected sizes and temps used:
Gene
Length (bp)
Tm Used (°C)
Optimal Temp (°C)
XylA
1053
50, 52, 54, 56
52
XylB
1101
52, 54, 56, 60
56
XylM
1110
50, 53, 56, 60
53
XylA + XylM
2163
50, 52, 56, 58
56
RFP
678
55, 56, 57, 58
56
PuP + RFP
678
55, 56, 57, 58
56
- Gel pictures (2)
- Extracted pSynth1-2, pSynth3, pSynth6 from Top10 and transformed into BL21 DE3. Growing transformed BL21 DE3 overnight
- Extracted plasmids were amplified by PCR and digested to confirm
- PCR via same Q5 protocol as above (100 ng ea. plasmid template)
- Digestion check
- All digested w/EcoRI and SpeI
- 1Ul each enzyme used; 100 ng template used
- Gel pic
- Repeated digestions with same protocol overnight and 300 ng template instead of 100 ng
June 23, 2016
- Gel picture from 6/23/16 digestion
- pSynth3, pSynth6 plasmids confirmed; need to re-extract pSynth1-2 and redo digestion
- Confirmed PCRs:
{XylR: 56 °C anneal, XylA + XylM: 56 °C, PuP + RFP: 59 °C, XylB: 56 °C} see 6/22 for temperature optimization experiment
- Gel pic: confirmation and pSynth5 plasmid confirmed
- pSynth5A plasmid confirmed
- Colony 1: all strains confirmed
June 24, 2016
- Gel picture
- pSynth1-2 plasmid confirmed
- Redoing PCRs for pSynth5 (incorrect primer?)
- Gel picture
June 25, 2016
- All pSynth5 stuff confirmed
June 26, 2016
- Digestions for plasmid construction:
- pSynth1-2: S,P
- pSynth3: X, P
- pSynth5: S, P
- pSynth6: X, P
- All in 2.1 Buffer
- 800 ng each template DNA in reactions
- 10 uL buffer
- 10 uL each enzyme
- 50 uL total reaction
- Started 9:35 AM, ran for 1 hour at 37 °C
- Gel picture
June 27, 2016
- The bands were too light to use on 6/26/16. Re-did the gel with 1500 ng.
- Need fresh pSynth1-2 (was fine on 6/24/16, but will re-extract to be safe)
- Gel picture
- Extracted and ligated XylA and XylB bands (circled on gel) into BL21 DE3 and Top10
- Transformed negative control (A only) into Top10
- 3:1 molar ratio, insert: B, used (B:A)
June 29, 2016
- Redoing ligation/transformation
- Digestion Protocol:
- pSynth1-2 with SPeI and PstI
- pSynth3 with XbaI and PstI
- 5 uL 2.1 Buffer
- 1 uL each enzyme
- 1500 ng DNA from plasmid
- add H2O to total 50 µL
- 37 °C water bath for 2 hours
- Gel picture
- Digestion checks pSynth5 with ~600 ng/reaction:
1) S
3) E
5) S,X
7) E,X
2) P
4) X
6) E,P
8) S,P
- Gel picture of digestions
- Gel picture of digestions troubleshoot
July 1, 2016
- Got 3 fresh colonies from BL21 DE3 and pSynth5 transformation
- PCR checked and digested fresh
- Plasmid extraction
- Expected:
XylR: 1701 bp
BB: ~2 kb
S + P: ~3900 bp
E + S: 1800 + 2000 = 3800 bp
- Gel picture New Digest and PCR check
- Gel picture pSB1K3 BB PCRs
July 4, 2016
- PCR for Kan BB for construction of pSynth5 B is good (colonies 1-3)
- Clean and concentrate failed
- Redo PCR
July 6, 2016
- Gel picture of pSB1K3
- Failed, redo
July 7, 2016
- Gel picture of pSB1K3
July 8, 2016
- Extracted fresh part from colonies
- PCR to get BB (temperature optimized as well)
- Gel picture
- Bands are at the wrong length, redoing PCR with Phusion
July 10, 2016
- Grew pSynth 1-2 and pSynth3 in BL21 DE3
- Plasmid extraction
- Digested as follows - 212 ng template each
- Gel picture pSynth 1, 2, 3 digestions
July 11, 2016
- Gel extraction performed (successful)
- Clean and concentrate in Dr. Dalhaimer's lab
- Gel picture Cm/Kan/Amp
- Kan BB unable to be amplified, will use Amp instead
- Amp BB isolated from the gel and will be used in Gibson + pSynth5; same for Cm BB
August 24, 2016
- Recording OD's and samples
- Strains:
1) pAMB – benzaldehyde production
2) pAM – benzyl alcohol production
3) wild-type – control
- Add 5 mM toluene or 5 mM benzyl alcohol
- Using pAMB + 5mM m-xylene and p-xylene
- Watching for blue pigments as by product
- Prepared solution (LB, Cm, toluene) 1-2 hours before t = 0
August 25, 2016
- Same experimental setup as done on 8/24/16
- Prepared solution (LB, Cm, IPTG) much earlier than t = 0
August 30, 2016
- Same experimental setup as done on 8/24/16 but with m–xylene and p–xylene
August 31, 2016
- pAMB "blue" test
- running 3 cultures with 25 mL LB + CM and 12.2 µL IPTG in a 250 mL flask:
1) pAMB
2) pAMB + toluene
3) pAMB + benzyl alcohol
September 2, 2016
- Prepared GC/MS samples
September 3, 2016
- Prepared all GC/MS samples at t = 0 and t = 5
- Diluted the blanks by 1/5
September 4, 2016
- p56 experiment – try denser cultures and less IPTG
- Inoculated p56 cells
September 8, 2016
- p56 experiment postponed – OD's grew uneven and subject to contamination
- pRFP (Kan) plates showing no growth – check with Dr. Trinh's pRFP strain
September 21, 2016
- p56 experiment did not work
- Toxicity study with gasoline performed in replicate
September 22, 2016
- Gasoline experiment inconclusive, but still have OD data
- Gasoline may have evaporated out
September 25, 2016
- GC/MS data looks bad – most compounds may have evaporated out
- Running evaporation tests – marked volume levels of the layers as a reference
