Week Seven

Monday, 7/28/14

• 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.

• Single-transformations run on each of the constructs used in the double-transformations:
  1. pMB001
  2. pMB002
  3. pMB003
  4. pMB004
  5. pMB005
  6. pMB006

• 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

Tuesday, 7/29/14

• 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

• 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.

• Miniprepped liquid cultures grown last night and shipped samples for sequencing.

Wednesday, 7/30/14

• Ran new PCR reactions with newly designed primers to extract backbones of pTG002/3/6.
• DpnI-digested (4 hrs) and PCR-purified the PCR products.
• Products were run on a gel to confirm their creation.
• Gibson assembled the backbones with their geneblock inserts
• Gibson assemblies were transformed into JM109, which were plated & incubated overnight.

• Western blots were run on both supernatant and cell lysate samples (6 concentrations of aTc, so 12 Western lanes for each system) from the 2 liquid cultures grown up last night. 3xFLAG-antibodies appear to mark proteins in the fsr supernatant and cell lysate lanes.

• 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.

Thursday, 7/31/14

• 5 colonies from each of the overnight transformations were picked (15 total), and we ran colony PCR (52°C annealing step) on them using the newly ordered sequencing primers.
• colony PCR products were run on a gel, showing that none of them contained the correct construct.
• 10 new colonies were picked and "colony-PCR-ed" (this time with a 47° annealing step). However, running the product on a gel showed these didn't work either.

• colony PCR was redone on the pMB001-006 transformations using the newly ordered primers. Still no binding.

• We used a gel extraction kit to extract the pAA009/010 backbone from the gel run yesterday.

• 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

Friday, 8/1/14

• To debug our colony PCRs from yesterday, we set up 8 colony-PCR reactions on a annealing temperature gradient, using E. coli containing pKS001 (the positive control in our earlier colPCRs) as the template.
  • Annealing temperature was allowed to range from 47°C to 54°C
  • Optimal annealing temperature for our new sequencing primers was determined to be around 48.6°C, with no binding beyond 52°C.
• To create a negative control for our Western blots, we obtained a pTet_GFP plasmid that had been assembled via Golden Braid assembly. This assembly product was transformed into JM109 and plated on a chloramphenicol plate.
  • We will eventually place a 3xFLAG behind the GFP in this plasmid to be used in a Western.
• In preparation for the LCMS run to detect the lam system's signalling peptide, we are going to remove the 3xFLAG from pTG004 (as it would be in any actual signal-reception system involving lam we would design):
  • 3xFLAG was PCRed out (linearizing the pTG004 plasmid) using 2 new primers we had designed with phosphorylated 5' ends. 50°C annealing step was used.
  • PCR-purified PCR product.
  • Around-the-horn ligation (to recircularize pTG004-without-FLAG) was run using Quick Ligation kit
  • Ligation product was transformed into JM109, plated on carbenicillin, and incubated.
  • Colony PCR (49°C annealing step) was then run on the 3 colonies that grew up.
  • A gel was run on the colony PCR products, revealing ____

• Still no success with the colony PCR.

• 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.