Dear research community,
I have met an interesting issue in a small research side project in which I am engineering some bacteria. For this though I would need to permeabilize the outer membrane of E.Coli in order to bring a bigger peptide (5kDa) or protein (72kDa) to the inner membrane. However, the cell should be preferably still alive (able to do gene expression).
I know that preparing a spheroplast might be one possibility. Does anyone have a good protocol for this? What would be a good control that I know that I have produced spheroplasts?
Do you have other suggestions then please let me know. :) Thank you...
Try using Tris Buffer with EDTA and sucrose to suspend the cells.
O.5M CaCl2 treatment permiabilize bacterial cells for the intake of plasmids.
@Ancy Thomas: I will not need to permeablize the entire cell but only the outer membrane ;)
An old article reported that EDTA-heat shock treatment of E coli cells resulted in temporary outer membrane gaps (Please refer http://www.ncbi.nlm.nih.gov/pmc/articles/PMC210360/pdf/jbacter00176-0036.pdf).
A recent work by Kikuchi et al reported E coli spheroplast preparation in detail (http://www.sciencedirect.com/science/article/pii/S2215017X15000260).
You can use surfactant at lower concentration to increase the permeability.
You can work on enhancing membrane fluidity by modifying membrane lipid composition.
Tris/EDTA treatment (Sumreet Singh Johar suggestion, possible also without sucrose) is O.K. for permeabilization of the OM to lipophilic compounds. But it makes only some phospholipid bilayer patches in the OM. It can help for your peptide, if it is amphiphilic. 70 kDa protein is more serious problem. You should make real spheroplasts (you can see them through microscope, they become spherical from bacillus form cells ) using lysozyme, but it will change the PM permeabilty and the viability of cells. I am not sure about the gene expression. We were able to get to the PM even bigger protein complexes and get reproduction of phage (see Poranen et al J Cell Biology, Volume 147, Number 3, 1999, 671–681) , but I am not sure what will be with your protein...
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