Team:Caltech/Week 11

August 22

Send BPA and degraded products to HPLC for analysis
Send 16s for sequencing using FWD and REV.
PCR'd R0040, K123001, B0014 and pSB3K3 for Gibson assembly of pNT002

Results

Plated 16s and WT-F87A cultures show many colonies
Mix 10% glycerol solution and place in 4&degC fridge for tomorrow's electrocompetence method
Overnight shake culture of MG-1655 experimental E. coli strain

August 23

Make experimental MG-1655 strain electrocompetent
Made 53 plates today!

• 24 LB-amp plates
• 16 LB-chlor plates
• 13 LB plates

PCR'd R0040 and pSB3K3 for Gibson assembly of pNT002
PCR'd the DDT gene with new primers to add stop codons and biobrick ends.
PCR'd the DDT gene with BamHI and XbaI sites and a C-terminus his tag for insertion into pET11-a. This will allow us to overexpress the protein for purification and characterization.
PCR'd WT-F87A with biobrick ends.
Dpn 1 digested the DDT and p450 PCRs overnight

August 24

Visited the San Jose Creek Water Reclamation Plant
Made more Gibson mix according to Joe's protocol.
Did double restriction digest of DDT gene and p450 PCRs with biobrick ends and pSB1C3 using EcoRI and PstI.
Did double restriction digest of DDT gene PCR with pET11-a insertion endings and pET11-a using BamHI and XbaI
Did CIP digest of pET11a and pSB1C3
Ligated DDT gene with biobrick ends into pSB1C3
Ligated p450 with biobrick ends into pSB1C3
Ligated DDT gene into pET11-a
Transformed all ligations into 50 ul aliquots of DH5a electrocompetent cells
Transformed 1 ul of 10 pg/ul pUC19 into 1 aliquot of DH5a electrocompetent cells
Started overnight of pSB1C3 so there is more DNA available to use as backbones for biobrick parts to send to the registry.

August 25

pSB1C3 concentration 118.0 ng/ul
Calculated transformation efficiency of DH5a electrocompetent cells is about 2x10^8 cfu/ug

Results