Template:Calendar

Team UT Knoxville

UTK
iGEM

2016

January
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February
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March
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April
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May
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June
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July
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August
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September
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October
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November
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December
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May 18, 2016

- 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 &degC 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 (&degC) Optimal Temp (&degC)
XylA 1053 50, 52, 54, 56 52
XylB 1101 52, 54, 56, 60 56
XylM 1110 50, 53, 56, 60 53
XylA &#43 XylM 2163 50, 52, 56, 58 56
RFP 678 55, 56, 57, 58 56
PuP &#43 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 &degC anneal, XylA + XylM: 56 &degC, PuP + RFP: 59 &degC, XylB: 56 &degC} 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 &degC
- 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 &microL
	-	37 &degC 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 &#61 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&#39s 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 &#43 pSynth5; same for Cm BB 
				

August 24, 2016

- Recording OD&#39s and samples
- Strains: 
	1)	pAMB &#150 benzaldehyde production
	2)	pAM &#150 benzyl alcohol production
	3)	wild-type &#150 control
- Add 5 mM toluene or 5 mM benzyl alcohol
- Using pAMB &#43 5mM m-xylene and p-xylene
- Watching for blue pigments as by product
- Prepared solution (LB, Cm, toluene) 1-2 hours before t &#61 0
				

August 25, 2016

- Same experimental setup as done on 8/24/16
- Prepared solution (LB, Cm, IPTG) much earlier than t &#61 0
				

August 30, 2016

- Same experimental setup as done on 8/24/16 but with m&#150xylene and p&#150xylene
				

August 31, 2016

- pAMB &quotblue&quot test
- running 3 cultures with 25 mL LB &#43 CM and 12.2 &microL 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 &#61 0 and t &#61 5
- Diluted the blanks by 1/5
				

September 4, 2016

- p56 experiment &#150 try denser cultures and less IPTG
- Inoculated p56 cells
				

September 8, 2016

- p56 experiment postponed &#150 OD&#39s grew uneven and subject to contamination
- pRFP (Kan) plates showing no growth &#150 check with Dr. Trinh&#39s 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 &#150 most compounds may have evaporated out
- Running evaporation tests &#150 marked volume levels of the layers as a reference