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 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

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

Wednesday, 7/9/14

Thursday, 7/10/14

Friday, 7/11/14