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1. Isolation of Escherichia coli or E. coli Co-transformants
- Prepare electro-competent cells of the appropriate E. coli strain to be transformed. Transfer to 1 ml of Luria-Bertani (LB) medium (Tryptone, Yeast Extract, sodium chloride or NaCl at 10, 5, and 10 g/L, respectively) a single colony of the strain of choice, and incubate at 37 °C under shaking conditions of 180 rpm. Dilute the pre-culture 1:500 in 25 ml of fresh LB medium and incubate the culture at 37 °C.
- At log-phase (0.6 O.D.), centrifuge the cell suspension at 5,000 x g for 20 min, and resuspend the pellet in 10%, v/v ice-cold sterile glycerol-water in half of the original culture volume. Repeat this step 4 times, halving the resuspension volume each time. Finally, resuspend the pellet in glycerol/water and divide the cell suspension into 50 μl aliquots. Store the aliquots at -80 °C up to 6 months.
- Dissolve the desired plasmid in sterile water supplemented with 0.5 mM ethylenediaminetetraacetic acid (EDTA). Thaw on ice an aliquot of electro-competent cells and mix with an appropriate amount (2.5-5 ng) of vector. Dispense the mixture into a 0.1 cm cuvette suitable for electroporation, and apply 1.8 kV.
- Immediately transfer the electroporated cells into 1 ml of super optimal broth with catabolite repression (SOC) medium (LB medium supplemented with 0.2% w/v glucose, 10 mM magnesium chloride or MgCl2, 2.5 mM potassium chloride or KCl), incubate for 1 hr under shaking, and finally transfer 100 μl aliquots to Petri dishes containing LB agar with the appropriate antibiotic. Incubate overnight (O/N) at 37 °C.
- Purify the transformants by streaking single colonies on Petri dishes.
- Prepare electro-competent cells of the primary transformants and repeat steps 1.1 to 1.5 to transform with further plasmids.
- Prepare glycerol stocks of the co-transformants. Transfer a single colony in LB supplemented with the appropriate antibiotics, incubate at 37 °C under shaking, and at log-phase (0.6 O.D.), centrifuge the cell suspension at 5,000 x g for 20 min. Resuspend the pellet in LB-antibiotics medium containing 15% v/v glycerol. Dispense in aliquots and store at – 80 °C.
2. Mutation Analysis of Populations Co-expressing the Wild-type ε Subunit of E. coli DNA Polymerase III and the Mutagenic εD12A Variant
- Transfer a single colony of E. coli TOP10 containing the pBAD-ε and the pGOOD1-εD12A vectors to 1 ml of LB-antibiotics medium. Incubate O/N at 37 °C.
- Dilute the pre-culture 1:250 in 3 flasks containing fresh medium (10 ml), add the appropriate inducers (arabinose, isopropyl β-d-1-thiogalactopyranoside (IPTG), or arabinose and IPTG, 1 mM each), and incubate at 37 °C for 8 hr. Prepare in parallel non-induced cultures.
- Collect aliquots of 1 ml, then dilute 1:500 in new flasks containing fresh medium (10 ml) supplemented or not with the appropriate inducers. Incubate O/N at 37 °C.
- Repeat steps 2.2 and 2.3 and collect aliquots of 1 ml.
- Determine the number of generations that occurred in each culture. Transfer on LB plates, 100 μl of appropriate serial dilutions of inoculum and culture. Incubate O/N at 37 °C. Count the colonies on the LB plates, and calculate the log of the number of cells present in the inoculum (logI) and in the culture at the end of growth (logC). To determine the number of generations, use the formula: (logC– logI)/0.301.
- Centrifuge at 5,000 x g for 20 min and resuspend the cells in 1 ml of 50 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 7.6, 50 mM NaCl. To permeabilize the cells, add 2-3 drops of chloroform and vortex for 20 seconds.
- Determine the β-glucosidase activity of each aliquot in a 96-well microplate, using p-nitrophenyl-β-D-glucopyranoside (PNPGluc) as substrate. Add to each well 100 µl of permeabilized cells and 100 µl of substrate (16 mg/ml stock solution in water or H2O). Take care to avoid air bubbles in the wells. Read the Absorbance at 420 nm, using a microplate reader and the appropriate filter.