Electro-transformation of Lactobacillus spp.

Overview

Instructions on how to prepare Lactobacillus plantarum competent cells before electrotransformation.

Materials

• MRS media
• Culture of L. plantarum cells
• MgCL₂ (10mM)
• sucrose
• glycerol
• Centrifuge capable of holding four 50mL centrifuge tubes.

Procedure

Day 1

1. Prepare the following:

• 25mL MRS media
• 25mL Treatment Media (MRS media with 4g glycine(4%) and 15g (1.8M) sucrose added).
• 50mL Water
• 50ml 50mM EDTA
• 50mL Electroporation Buffer (0.9M Sucrose, 10%(v/v) glycerol)

1. Cap the flasks with foil and autoclave.
2. Once the MRS has cooled, inoculate the flask without glycine with L.plantarum culture and grow overnight at 30°C.

Day 2

1. Put the buffers on ice and pre-chill the centrifuge.
2. Add the 25mL Treatment Media to the overnight culture.
3. Incubate cells for 1-1.5 hr at 30°C.
4. Divide culture into two 50mL centrifuge tubes.
5. Centrifuge for 2 minutes at 5000g.
6. Pour off supernatant and resuspend pellet in 10mL ice-cold water.
7. Centrifuge for 2 minutes at 5000g.
8. Pour off supernatant and resuspend pellet in 10mL ice-cold water.
9. Centrifuge for 2 minutes at 5000g.
10. Pour off supernatant and resuspend pellet in 25mL EDTA solution.
11. Let cell suspension sit on ice for 30 mins.
12. Centrifuge for 2 minutes at 5000g.
13. Pour off supernatant and resuspend pellet in 10mL ice-cold Electroporation Buffer.
14. Centrifuge for 2 minutes at 5000g or until supernatant is clear.
15. Pour off supernatant and resuspend cells in 500μL ice-cold Electroporation Buffer.
16. Dispense into 100μL aliquots.
17. Store at -20°C for use that day.

Notes

All questions, input and feedback are welcome!

1. Centrifugation at 4000 rpm for 2 minutes was sufficient to pellet the competent cells to give a clear supernatant.

References

Relevant Papers and Books

Alegre et al (FEMS Microbiology Letters 241 (2004) 73-77)

Contact

• morto077@uottawa.ca

or instead, discuss this protocol. -->