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 Seven |
Monday, 7/28/14
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Export Systems
- Ran colony PCR on pTG002/3/6-transformed JM109 cultures grown up over the weekend. Bad results for all of them
- Created and ordered new primers that will hopefully avoid secondary-structure-formation when the exonuclease attacks the 5' end during Gibson assembly.
lamBCDA & fsrABC Reception Systems
- Single-transformations run on each of the constructs used in the double-transformations:
- pMB001
- pMB002
- pMB003
- pMB004
- pMB005
- pMB006
agrBCDA Reception System and Combinatorial Promoters
- Colonies were picked from pAA008 transformations incubated over the weekend, as well as the pAA009 & pAA010 transformations, which had been set up last Thursday.
- Set up liquid cultures of the colonies to grow overnight in preparation for miniprep and sequencing tomorrow
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Tuesday, 7/29/14
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Export Systems
- Ran Gibson assembly products on a gel. Results inconclusive
- Started 3 mL liquid cultures of bacteria (with pTG004 & pTG005) with varying levels of aTc induction:
- 0 nM, 50 nM, 150 nM, 250 nM, 350 nM, & 450 nM
lamBCDA & fsrABC Reception Systems
- Colony PCR was run on several of the colonies that emerged from the single-transformations last night
- Gel was run, but wrong primers were used. New primers for sequencing/colPCR were ordered, and colPCR will be redone tomorrow.
agrBCDA Reception System and Combinatorial Promoters
- Miniprepped liquid cultures grown last night and shipped samples for sequencing.
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Wednesday, 7/30/14
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Export Systems
lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters
- Analyzed sequencing results:
- pAA008 successfully constructed
- Minipreps supposedly containing pAA009 & pAA010 instead contain pKS001 backbone template
- We redid PCR of the pAA009/010 backbone
- DpnI digestion for 2 hours, followed by PCR purification. The purified product was then run on a gel.
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Thursday, 7/31/14
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Export Systems
lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters
agrBCDA system:
- We used a gel extraction kit to extract the pAA009/010 backbone from the gel run yesterday.
Combinatorial Promoters:
- PCRed the plasmid backbone and combinatorial promoter parts of pAA002.
- DpnI digested the PCR products
- Ran a gel on the products to confirm sequences were of the correct length
- PCR-purified the DpnI-digested products
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Friday, 8/1/14
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Export Systems
lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters
- Did a Gibson assembly of pAA002 (containing a combinatorial promoter), pAA009, and pAA010 (containing agr reception system), using PCRed/gel-extracted (respectively) products from yesterday.
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