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. Add the 25mL MRS plus Glycine.
2. Incubate cells for 3-4 hrs at 30°C until OD₆₀₀ is approximately 0.85.
3. Put the buffers on ice and pre-chill the centrifuge.
4. Pour Cultures into 50mL centrifuge tubes.
5. Centrifuge for 2 minutes at 5000g or until supernatant is clear.
6. Pour off supernatant and resuspend pellet in 10mL ice-cold Wash Buffer.
7. Centrifuge for 2 minutes at 5000g or until supernatant is clear.
8. Pour off supernatant and resuspend pellet in 10mL ice-cold Wash Buffer.
9. Centrifuge for 2 minutes at 5000g or until supernatant is clear.
10. Pour off supernatant and resuspend pellet in 10mL ice-cold Electroporation Buffer.
11. Centrifuge for 2 minutes at 5000g or until supernatant is clear.
12. Pour off supernatant and resuspend cells in 1-2mL ice-cold Electroporation Buffer.
13. Can then be stored in an ice bath for no more than 4hrs before use.
14. Store in aliquots and store in a -80°C freezer.

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. -->