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 Four |
Monday, 7/7/14
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Export Systems
- PCR extracted vector backbone from pKS001 with Biobrick prefix & suffix overhangs
- PCR purification of PCR product and then 1 hour incubation at 37°C with DPN1 enzyme to remove any remaining sticky ends
- PCR purification of DPN1-digested product
- Ran a gel that confirmed presence of ~3000bp-long DNA fragment in both DPN1-digested and undigested PCR products
- Gibson Assembly of the PCR fragments with the geneblocks ordered 2 weeks ago to form the 4 exportDomain-comX-mNeonGreen constructs (incorporating GeneIII, OmpA, PelB, and TorA as the export domains in the N-terminus) and the 1 comX-mNeonGreen-HlyA construct, all in the vector backbone
- Transformation of assemblies into JM109. Plated on ampicillin plates and incubated at 37°C overnight
Combinatorial Promoters
- Set up liquid cultures of the colonies that yielded promising results in the colony PCR conducted last Thursday; cultures incubated overnight
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Tuesday, 7/8/14
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Export Systems
- Picked 4 colonies per plate incubated last night and resuspended the bacteria in 10 uL H2O (20 colonies picked total, 4 representing each of the 5 export domains)
- Colony PCR was then run on 1 uL samples of the resuspensions
- Unfortunately, PCR products showed no DNA constructs on running through a gel
- Picked 10 resuspensions (2 of each export domain) and used them to inoculate 5 mL liquid cultures, which were placed in the 37°C shaker overnight
Combinatorial Promoters
- Miniprepped liquid cultures grown up last night to extract plasmids
- Sent in the resulting plasmids for sequencing
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Wednesday, 7/9/14
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Export Systems
- Discovered that the reverse primer used in colony PCR last night had no binding site on the plasmids we had assembled (pTG001 constructs)
- Miniprepped liquid cultures grown up last night to extract plasmids
- Not all bacteria were able to be resuspended after pelleting, so resulting DNA yields for 6 of the 10 liquid cultures grown were very low (10-25 ng/uL)
Combinatorial Promoters
- Analyzed sequencing results and grew more liquid cultures of the colonies containing properly assembled plasmids
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Thursday, 7/10/14
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Friday, 7/11/14
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