Danuška Horáková

The effect of plasmid DNA sizes and other factors on electrotransformation of Escherichia coli JM109

Abstract The plasmid size can be an important factor in electrotransformations. We have examined bacterial electroporation with a specific interest in the transformation of plasmids with different sizes of their molecules. We used plasmids pUC19, pBR322 and pPP4. Transformation efficiency drops with increasing size of the DNA. We achieved with plasmid pUC19 a 81% frequency of transformation. The optimal field strength decreases with increasing size of the plasmid. Not only large sized plasmids but also large DNA concentrations lead to a reduced survival rate of the Escherichia coli JM109 cells.

The influence of the growth phase of enteric bacteria on electrotransformation with plasmid DNA

Salmonella typhimurium LB5000 andEscherichia coli JM109 were transformed by electroporation. In accordance with the chemical transformation methods, the growth phase of these electrocompetent bacteria had a strong impact on transformation efficiency. Survival of bacteria, after the high-voltage electrical pulse was also influenced by the growth phase. Both bacterial species were most successfully electrotransformed when microbial cells were harvested at the late lag phase. The second optimum for transformation reachedE. coli cells in the mid-exponential andS. typhimurium cells in the late exponential phase. Transformation efficiencies ranged from 3.4×104 to 2.7×105 transformants per μg DNA in the case ofS. typhimurium and from 2.8 × 102 to 8.8×105 transformants per μg DNA in the case ofE. coli. Survival of cells after the electrical pulse in late lag and late exponential phases was about 20% higher than during other phases of growth. Preparing electrocompetent cells from later phases of their growth is more useful for practice, because it provides more biomass with good yield of transformants.