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Abstract
Escherichia coli are non-competent, gram-negative cells possessing an outer membrane (OM), in addition to a cell wall and cytoplasmic membrane, as a barrier to transformation by the uptake of environmental DNA. As it has been reported that spheroplasts may be transformable at 100-fold higher efficiency than control E. coli DH5 α using a modified version of the Hanahan protocol, this study was aimed at determining the effect of the cell wall and OM in transformation, in the absence of competence inducing divalent cations. Spheroplasts induced from an exponentially growing culture were subjected to both heatshock and electrotransformation methods using pUC8 plasmid DNA (conferring ampicillin resistance to transformed cells), and were assessed for survival rates and transformation efficiencies. Here, we report that while heat-shocked spheroplasts had survival rates 46 to 71% of control cells, divalent cations play a critical role in transformation, and thus no transformants were produced. We also found that spheroplasts survived electroporation at rates 36 to 51% of non-spheroplasted cells, but the transformation efficiency was questionable.