Bacterial Recombinant Protein Expression

The pBAD plasmids are pBR322-derived expression vectors designed for regulated, secreted recombinant protein expression and purification in E. coli.

Steps

1. For each transformant or control, inoculate 2 mL of SOB or LB containing 50 µg/mL ampicillin with a single recombinant E. coli colony. Note: If you are using LMG194 as a host, use RM medium containing glucose and 50-100 µg/mL ampicillin at all steps.
2. Grow overnight at 37°C with shaking (225–250 rpm) to OD 600 = 1–2.
3. The next day, label five tubes 1 through 5 and add 10 mL of medium containing 50 µg/mL ampicillin.
4. Inoculate each tube with 0.1 mL of the overnight culture.
5. Grow the cultures at 37°C with vigorous shaking to an OD 600 = ~0.5 (the cells should be in mid-log phase)(~5 hours).
6. While the cells are growing, prepare four 10-fold serial dilutions of 20% L-arabinose with sterile water (e.g. 2%, 0.2%, 0.02%, and 0.002%).
7. Remove a 1 mL aliquot of cells from each tube, centrifuge at maximum speed in a microcentrifuge for 30 seconds, and aspirate the supernatant.
8. Freeze the cell pellet at –20°C. This is the zero time point sample.
9. Add L-arabinose to the five 10 mL cultures as follows:

1. Grow at 20-25°C with shaking overnight.
2. Take 1 mL samples and treat as in Step 7 and 8.