Transform Lactobacillus

[butterfly_2010]

Dear Forum experts,

I tried to transform Lactobacillus delbrueckii by electroporation using the following protocol (see below). It worked nicely before but it suddenly stopped working recently. Basically, I did not get any colony. I repeated this experiment several times without any success. I just couldn’t figure out what went wrong. Could you please help?
Thank you!

PS: My protocol:
1. Inoculate 50 ml MRS in a 50-ml falcon tube using 0.5 ml of an overnight starter culture and incubate at 37°C until it reaches an OD600 = 0.5 – 0.8.

2. Centrifuge culture in the 50-ml falcon tube at 4000 rpm for 10 min at 4ºC. Discard the supernatant and resuspend the pellet with 10 ml of cold electroporation buffer (0.286 M Sucrose, 1 mM MgCl2).

3. Centrifuge the cells at 4000 rpm, 4ºC, 10 min; discard the supernatant. Resuspend the cells in 10 ml of electroporation buffer.

4. Repeat step 3 once.

5. Resuspend cells in 0.5 mL electroporation buffer (concentrate 100 fold).

6. Transfer 0.2 mL cells to an eppendorf tube and add 0.5 μg of DNA; mix and transfer the cells to a cold 0.2-cm cuvette.

7. Electroporate at 2.45 kV, 25 μFD, 200Ω using Gene Pulser (Bio-Rad) (Note: I also tried 1.5 kV and 2.0 kV. They didn’t work either).

8. Transfer the cells to 1.0 mL MRS immediately and incubate overnight at 37°C.

9. Plate 50 μl of transformed cells onto an MRS plate with 50 μg/ml of erythromycin.