Production of a new DNA vehicle for gene transfer using site-specific recombination

Supercoiled DNA molecules, minicircles, were produced by in vivo site-specific recombination. They contained exclusively the desired excisable fragment. Recombination was driven by bacteriophage lambda integrase from a plasmid substrate containing the attP and attB recombination sites in the same orientation. Conditions for minicircle production within the lysogen Escherichia coli D1210HP were optimised. Up to 1.5 mg minicircles could be produced per litre bacterial culture, and the remaining, unrecombined plasmid comprised less than about 15% of the minicircle produced. However minicircle multimers were also produced, and comprised up to 30% of all minicircles synthesised. The par ABCDE' locus from plasmid RK2 was introduced into the minicircle fragment, resulting in minicircle dimers being reduced to less than 2% of all minicircles. The par A gene encodes a resolvase that catalyses recombination at the multimer resolution site in the parABCDE' locus. Minicircle multimers were also resolved when par A was introduced downstream from the integrase gene of the lambda pL transcript in D1210HP together with a multimer resolution site carried by the minicircle fragment.