Team:Caltech/Week 12

August 28

Made R0010-MG1655 and J23119-MG1655 plates and overnights
Continued growing biofilm in flask with glass beads; added fresh amphicillin
Mixed more X-gal and IPTG solution at 20mg/mL and .1M concentrations, respectively
Attempted to induce R0010 overnights with IPTG to cleave X-gal; J23119 to cleave X-gal without inducing

Results

J23119-MG1655 grew, but the R0010-MG1655 did not
R0010 did not cleave X-gal even with a high concentration of both IPTG and X-gal
J23119 cultures turned blue with cleaved X-gal after about 4 minutes

August 29

R0010 overnight culture did not grow
Redo p450 experiments in MeOH in order to send to electrospray
Attempt to measure J23119-X-gal reaction with spectrophotometer

Results

J23119 culture was too thick and not in exponential growth phase; redo

August 30

HPLC of yesterday's p450 reaction and purification
Overnights of DDT-pET, WT-F87A, and lacZ colonies for sequencing, BioBrick submission, spectrophotometer experiments, etc.
Make M9 media for suspending X-gal for biofilm column experiments
Obtain pumps, tubing, and clear columns for biofilm-X-gal experiment

Results

HPLC results revealed too much noise; chemicals at too low concentration

August 31

Miniprep and sequencing of DDT-pET with T7 promoter and terminator, as well as WT-F87A using internal primers
Scale up p450 degradation reaction to 500uL for HPLC

Results

R0010 glycerol stocks are not growing overnights

September 1

Sequencing of DDT-pET revealed correct results
Attempt at bisdB PCR with ultrimers was not successful
Made 250mL SOC media

Results

HPLC did not yield expected results

September 2

pSB3k3 vector transformed into XL10-gold strain
Plate glycerol stocks of K123001 and pNT001