Team:MIT/Transforming Lactococcus lactis


 * Pretreatment of L. lactis with lithium acetate and/or dithiothreitol was as follows: 109 cells were suspended at room temperature for 30 min in 8 ml of 100 mM LiAc, 10 mM DTT, 0.6 M sucrose, and 10 mM Tris-HCl, pH 7.5. Following treatment, the cells were pelleted, resuspended in 1.5 ml microcentrifuge tube, and washed as described above.


 * Electroporation: Overnight cultures of L. lactis of both strains grown at 37°C MRS broth were diluted 1:12.5 in 25 ml of MRS. Cells were harvested by centrifugation at 10,000 g for 10 min when the optical density at 660 nm was between 0.26 and 0.38. The cells were washed sequentially with the following ice-cold solutions by alternate centrifugation and resuspension: bidistilled water 2.0 ml, bidistilled water 1 ml, 50 mmol/l EDTA 1 ml, bidistilled water 1 ml, 0.3 mol/l sucrose 1 ml, 0.3 mol/l sucrose 0.3 ml. After the final suspension, the cells were immediately electroporated at a concentration of 1010 cells/ml, using an electroporator with pulse controller (Electro Cell Manipulator™ 600, BTX, USA). Electroporation was performed by a single pulse at 2.5 kV (E = 12.4 kV/cm), 200 Ω, and 25 μF (corresponding to pulse length of 4.6 ms), in 2 mm disposable electroporation cuvettes, using 1 μg of purified plasmid DNA. The cell suspension was diluted immediately by the addition of 5 ml MRS broth supplemented with 10 μg/ml CM and incubated for 2 h at 37°C before being placed on MRS agar supplemented with CM (10 μg/ml). Transformation was confirmed by selection in CM containing medium and plasmid analysis.