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1. Effect of Chaperone HdeB Overexpression on E. coli Survival under Acid Stress
NOTE: E. coli MG1655 genomic DNA was isolated using standard protocol.
- Amplify chaperone hdeB from E. coli MG1655 by PCR using primers hdeB-BamHI-rev GGT GGT CTG GGA TCC TTA ATT CGG CAA GTC ATT and hdeB-EcoRI-fw GGT GCC GAA TTC AGG AGG CGC ATG AAT ATT TCA TCT CTC C.
- Set up the PCR reaction in 50 µl as follows: 10 µl 5x polymerase buffer, 200 µM dNTPs, 0.5 µM primer JUD2, 0.5 µM primer JUD5, 150 ng genomic DNA MG1655, 0.5 µl DNA polymerase, add ddH2O to 50 µl.
- Perform amplification of hdeB as follows: Step 1: 5 min at 95 °C, 1 cycle; step 2: 30 sec at 95 °C, 30 sec at 55 °C, 30 sec at 72 °C, 40 cycles; Step 3: 10 min at 72 °C.
- Clone resulting PCR fragment into the EcoRI and BamHI sites of plasmid pBAD18 using standard methods for restriction site cloning. Purify the plasmid using a plasmid purification kit according to manufacturer's instructions. Verify the resulting plasmid by sequencing.
- Transform the plasmid expressing HdeB or the empty vector control pBAD18 into strain BB7224 (ΔrpoH) (genotype: F-, λ-, e14-, [araD139]B/r Δ(argF-lac)169 flhD5301 Δ(fruK-yeiR)725(fruA25) relA1 rpsL150(SmR) rbsR22 Δ(fimB-fimE)632(::IS1) ptsF25 zhf::Tn10(TcS) suhX401 deoC1 araD+ rpoH::kan+) using chemically competent cells.
NOTE: This strain is temperature-sensitive. - After 45 sec heat-shock at 42 °C and prior to the plating, incubate cells at 30 °C and 200 rpm. Perform single colony streak-outs of the positive clones and incubate overnight at 30 °C. Prepare an overnight culture in 50 ml LBAmp and cultivate the cells at 200 rpm and 30 °C.
- Dilute overnight cultures 40-fold into 25 ml LBAmp and grow the bacteria in the presence of 0.5% arabinose (Ara) at 30 °C and 200 rpm to an O.D.600nm = 1.0 to induce HdeB protein expression.
- For the pH shift experiments, use LBAmp+Ara to dilute the cells to an O.D.600nm of 0.5 and adjust to the respective pH values (here: pH 2.0, pH 3.0, and pH 4.0) by adding appropriate volumes of 5 M HCl.
- After the indicated time points (pH 2, 1 min; pH 3, 2.5 min; pH 4, 30 min), neutralize the cultures by addition of the appropriate volumes of 5 M NaOH.
- Monitor the growth of the neutralized cultures in liquid culture for 12 hr at 30 °C using O.D. measurements.