I am working with the E. coli strain BAS901, which has a mutation in lptD rendering the cells hyperpermeable via defective LPS assembly. I need to introduce a plasmid into this strain. Unfortunately, the standard protocol I use to make electrocompetent E. coli cells is not working. I've even tried preparing DH5alpha electrocomp cells at the exact same time, using the same procedure. The DH5alpha cells came out quite competent, and the BAS901 strain is totally unable to take up the same plasmid.
My procedure for making electrocompetent cells basically involves growing cells near and OD600 of 0.4, spinning down, and washing the pellet several times with distilled water. Finally I resuspend in a small amount of water with 10% glycerol. The entire procedure is done on ice.
Is it possible that, because BAS901 is a hyperpermeable strain to begin with, I'll need a different procedure for creating competent cells or performing the electroporation itself?
Hi,
I would also suggest to provide more results of electroporation of BAS901 vis-avis DH5alpha,to know if it is not all competent or getting killed at that voltage. I would prefer to vary the field strength for this strain. Previous studies on E. coli strains have shown that the Field strength can vary from 10,000 V/cm to 20,000 V/cm. So better to vary and see the response. In addition, keep a balance of % cell survival and %cell transformed. A trick I can suggest (it may or may not work) is to keep the time-constant lee for your hyperpermeable strain , to reduce the diffusion of the plasmid out from the cell (if this is also playing some role in low or no transformation)
best regards
You might try simple chemical competence such as any of the CaCl2 instead of electroporation, or the PEG/DMSO method (http://www.pnas.org/content/86/7/2172.full.pdf).
The other possibility would be to have a strain already carrying the plasmid and then use P1 transduction to move the mutation into that strain.
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