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Abstract
The Hanahan protocol is widely used to transform bacterial cells with a plasmid of interest. However, little is known about the mechanism by which bacterial cells, particularly Escherichia coli, take up these plasmids. It was hypothesized that the PitA phosphate uptake 2+ system, which has been shown to transport divalent metal cations including Ca, may be 2+ involved in the Ca -mediated plasmid uptake in the Hanahan protocol. The model on which 2+ this hypothesis is based involves the binding of plasmid DNA by Ca, followed by the 2+ localization of the Ca -plasmid complex to the PitA system and subsequent complex uptake + into the cell. In this study, the transformation efficiencies of a pitA mutant and a pitA wild type control were compared. A modified version of the Hanahan protocol was used to transform these strains with plasmid pBR322. Transformation efficiencies were determined by comparing ampicillin-selective with non-selective plate counts. This study indicated that the presence or absence of pitA has little impact on plasmid uptake in these transformations. Future studies on the basis of this study’s data must be done at plasmid-saturating con ditions for both the mutant and parental strains in order to support these results.