Week Four

Monday, 7/7/14

• 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 unwanted fragments
• 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

• Set up liquid cultures of the colonies that yielded promising results in the colony PCR conducted last Thursday; cultures incubated overnight

Tuesday, 7/8/14

• 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

• Miniprepped liquid cultures grown up last night to extract plasmids
• Sent in the resulting plasmids for sequencing

Wednesday, 7/9/14

• Discovered that the reverse primer used in colony PCR last night had no binding site on the plasmids we had assembled (pTG001 constructs), explaining the absence of any bands on the gels used for colony PCR
• 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)
• Decided to scrap the last 2 day's experiments and ordered proper forward and reverse primers for colony PCR of pTG001 plasmids to be done tomorrow

• Analyzed sequencing results and grew more overnight liquid cultures of the colonies containing properly assembled plasmids

Thursday, 7/10/14

• Redo of Colony PCR on the same colonies picked on July 8 (colonies had been streaked on separate plates to "save" them), but this time with new primers (expdom_seq_F & expdom_seq_R), which arrived today:
  • Still no bands visible. Concluded too much bacteria used in colony PCRs.

• Miniprepped 2 liquid cultures of bacteria, one containing plasmid pWW1521 (encoding a response regulator) and the other containing pWW1523 (encoding the response regulator linked to a SH3 scaffold peptide). Both express kanamycin resistance.
• 2 cultures of DH5α were transformed, plated on Kan+Cam plates, and then incubated overnight:
  • Transformed with pWW1521 & pWW2149 (plasmid encoding a receptor linked to the SH3 scaffold receptacle; expresses chloramphenicol resistance)
  • Transformed with pWW1523 & pWW2149

• PCR amplification of backbone and other parts to be used in pAA003 plasmid (containing the pBAD-pTet combinatorial promoter)

Friday, 7/11/14

• Picked 8 new GeneIII colonies and 8 new Hyl colonies (in addition to the pKS001-expressing positive control) to run colony PCR on.
  • While a band appeared at roughly 1500bp, as expected for the positive controls, no bands over a few hundred bps long were found for any of the experimental runs

• Picked colonies from overnight plates and streaked them out on new Kan+Cam plates to grow on the benchtop over the weekend.

• Ran gel on Thursday's PCR products
• DpnI digestion of PCR products to remove sticky ends
• Gibson assembly of pAA003 plasmid