Week Six

Monday, 7/21/14

• 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

• 4 cultures of JM109 were transformed, plated on Kan + Cm plates, and incubated overnight at 37°C:
  1. pMB001 and pMB003
  2. pMB002 and pMB003
  3. pMB004 and pMB006
  4. pMB005 and pMB006

• Ran a gel of the PCR product created on Friday (pAA009/10 backbone). Results were negative.
• Tried PCR again using lower annealing temperatures. Results were negative again.

• Set up overnight culture of jwAA001 strain to do more experiments tomorrow

Tuesday, 7/22/14

• Did colony PCR on transformed colonies incubated last night and ran a gel on the products.
• Several lanes showed multiple bands, potentially indicating nonspecific primer binding, 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

• 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

• ([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)

• 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

Wednesday, 7/23/14

• Miniprepped the 7 overnight liquid cultures and shipped them out for sequencing

• 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)

• Sequencing results confirm one colony was transformed with pAA003 (with D46 combinatorial promoter).
• Liquid culture of that colony was grown up overnight.

• 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.

Thursday, 7/24/14

• Sequencing results came in and were analyzed, revealing that all our colonies contained original pKS001 template
• A gel was run on the Gibson assemblies to troubleshoot, with the pKS001 template in a separate lane. Unfortunately, the pKS001 was at too low a concentration to detect on the gel, but it is highly suspected that the single band that did show up in the Gibson lanes corresponds to the pKS001 plasmid.

• Double-transformed yesterday's Gibson assemblies into JM109:
  1. pMB001 and pMB003
  2. pMB002 and pMB003
  3. pMB004 and pMB006
  4. pMB005 and pMB006

• Transformations from last night (pAA008-pAA010) failed.
• Tried single transformations (instead of double) of the Gibson products to troubleshoot the transformation. Transformed cells were plated and incubated overnight.

• ([aTc] in ng/ml, % arabinose): (0,0); (25,0); (50,0); (1000,0); (0,0.001); (25,0.001); (50,0.001); (1000,0.001); (0,0.01); (25,0.01); (50,0.01); (1000,0.01); (0,0.1); (25,0.1); (50,0.1); (1000,0.1)

Friday, 7/25/14

• PCR on pKS001 to obtain backbones.
• DpnI digestion using 1 μL enzyme for 4 hours. A different stock was used.
• DpnI digest was PCR-purified, and a gel was run on processed product & unprocessed product, confirming presence of pTG002 and pTG003 backbones.
• Gibson assembly run to assemble backbones with gblock inserts
• Gibson products were transformed into JM109 (added a 1 hour SOC incubation step post-introduction of plasmid). The JM109 cells were then plated & incubated overnight.

• 6 stocks of DH5α-Z1 were transformed with the 2 copies of pTG004 (lam-system) and 4 copies of pTG005 (fsr system).
• After 90 minutes of SOC incubation, the cultures were plated on CARB plates over the weekend.

• Last night's 4 double-transformations failed again.
• Redid the 4 double-transformations but using 3 μL of each plasmid instead of 1 μL. Plates were left on the tabletop over the weekend.

• Gibson assembly of pAA008 was repeated.
• Gibson product was transformed into JM109
• Ran a gel of Gibson products from today and yesterday (2 reactions for each of pAA008-10). Gels indicate nothing wrong with transformation--only 1 band appears in each lane at desired length.

• It appears that the pBAD-pTet promoter is dependent on only aTc concentration, since varying arabinose concentration doesn't affect gene expression very significantly.