bacterial transfeomation - (Nov/02/2011 )
I have a question about chemical transformation by cacl2/heat shock method mechanism. it is said that the 42 degree heat shock creates pores in bacterial cell wall that leads to plasmid entry into periplasm. please let me know how is it possible that a simple temperature shift break tick layer of peptidoglycan in outermenbrane. is there any other mechanism?
Cells are made competence by re-suspension in CaCl2 solution at 0°C.
...under these conditions, the Ca ions is create pores in the membrane, assist binding of the DNA to the cell membrane and mask the negative charge on the DNA ...allowing it’s passage through the hydrophobic cell membrane. The DNA is forced into the cells by applying a short 42°C heat shock, which results in a thermal current that sweeps the DNA into the cells.
And with respect to the Peptidoglycan cell wall, in gram negative bacteria like E. coli, the PPG is not very thick and is not as extensively crosslinked as the PPG in Gram positives. The cell wall/outer membrane structures that define gram negatives are part of what makes DNA tranformation work. Ask anyone who works with gram positive bacteria, and they will tell you that transforming a gram positive is way more difficult than E. coli.