Nov 26, 2020

Public workspaceEnterococcus faecalis protoplast generation

This protocol is a draft, published without a DOI.
  • 1In-house protocol
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Protocol Citation: Elizabeth Fozo 2020. Enterococcus faecalis protoplast generation. protocols.io https://protocols.io/view/enterococcus-faecalis-protoplast-generation-bp24mqgw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 25, 2020
Last Modified: November 26, 2020
Protocol Integer ID: 44860
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Materials
Required media
  • BHI
  • Isotonic solution – 20% sucrose, 0.145M NaCl, 50mM Tris-HCl
Before start
Required media
  • BHI
  • Isotonic solution – 20% sucrose, 0.145M NaCl, 50mM Tris-HCl
Protocol
Protocol
Grow OG1RF in BHI + condition until OD600 0.3. This works for as little as 10mls of culture.
Note
Growing in 2% glycine will reduce “gluing” of cells, but massively increase generation time.

Spin cells down and resuspend in half volume isotonic solution.
Add 2mg/ml lysozyme and incubate for 60 minutes. More time may be required.
Check for wall removal using Gram stain. (Do not move on until protoplast confirmation)
Spin cells down and wash with isotonic solution, resuspending again in isotonic solution in half the volume of the original culture.
Note
When resuspending cells after lysozyme treatment, pipette mix and refrain from over-vortexing as this will kill protoplast cells.