Overview
Week 1
Week 2
Week 3
Week 4
Week 5
Week 6
Week 7
Week 8
Week 9
Week 10
Week 11
Week 12
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Week Six |
Monday, 7/21/14
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3 of our 5 constructs were not properly cloned, so we are going to redo them:
- PCRed out backbones for pTG002, pTG003, and pTG006 with overhangs
- PCR product was then DpnI digested and PCR-purified. A gel was run with processed and unprocessed product to verify backbone creation
- Gibson assembly of pTG002, pTG003, and pTG006 (pTG006 had 2 reactions: 1 with the backbone created last week, since it had been verified to work for cloning pTG005, and another with backbone created today)
- Assemblies were transformed into JM109, plated on carbenicillin plates, and incubated overnight
lamBCDA & fsrABC Reception Systems
- 4 cultures of JM109 were transformed, plated on Kan + Cm plates, and incubated overnight at 37°C:
- pMB001 and pMB003
- pMB002 and pMB003
- pMB004 and pMB006
- pMB005 and pMB006
agrBCDA Reception System and Combinatorial Promoters
For the agrBCDA System, we troubleshot our problems creating the pAA009/10 vector backbone:
- Ran a gel of the PCR product created on Friday (pAA009/10 backbone). Results were negative.
- Tried PCR again with lower annealing temperatures [(what temps?)]. Results were negative again.
For the combinatorial promoters:
- Set up overnight culture of jwAA001 strain to do more experiments tomorrow
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Tuesday, 7/22/14
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- Did colony PCR on transformed colonies incubated last night and ran a gel on the products.
- Several lanes showed multiple bands [link to picture of gel], so we did a gradient PCR, varying the annealing step's temperature from 47°C to 55°C., and then ran a gel on the products
- Liquid cultures of 7 of the 10 picked colonies were prepared for overnight incubation
lamBCDA & fsrABC Reception Systems
- Transformations yesterday failed (no colonies sprung up overnight).
- A gel was run on the products of Gibson assembly to try to debug the cloning. Gel yielded no bands in any lanes, indicating that there were no Gibson assembly products to begin with.
- PCRed the backbone again, trying to get a higher DNA concentration for the next Gibson assembly
agrBCDA Reception System and Combinatorial Promoters
Retested jwAA001 system in triplicates:
- ([IPTG] in μM, [aTC] in ng/mL):(0,0); (5,0); (50,0); (500,0); (0,25); (5,25); (50,25); (500,25); (0,50); (5,50); (50,50); (500,50); (0,100); (5,100); (50,100); (500,100)
For agrBCDA system, we continued to try to create the pAA009 backbone:
- Redid PCR of pAA009 backbone, adding DMSO to the master mix to unravel any secondary structures. Also both pWW1523 and pWW1521 were used as the template DNA (since they should yield the same product).
- A gel was run on the product and suggested successful PCRing of the backbone
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Wednesday, 7/23/14
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Export Systems
- Miniprepped the 7 overnight liquid cultures and shipped them out for sequencing
lamBCDA & fsrABC Reception Systems
- PCR product created yesterday (backbone) was DpnI digested and then PCR purified.
- Gel was run on purified product, confirming its creation and existence.
- Redo of Gibson assembly with the new backbone (at higher concentration)
agrBCDA Reception System and Combinatorial Promoters
Combinatorial Promoters:
- Sequencing results confirm one colony was transformed with pAA003 (with D46 combinatorial promoter).
- Liquid culture of that colony was grown up overnight.
AgrBCDA system:
- Gibson assembly of pAA008-pAA010 using pAA009 backbone PCRed out yesterday and pAA008 backbone PCRed out Friday.
- Transformation of JM109 cells with Gibson products. Plated & then incubated.
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Thursday, 7/24/14
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Export Systems
lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters
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Friday, 7/25/14
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Export Systems
lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters
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