One-Step Bacterial Artificial Chromosome (BAC) Modification: Transfer of the Reporter Vector into BAC/RecA Cells and Selection of Co-Integrates.

ABSTRACT

In one-step bacterial artificial chromosome (BAC) modification, recombination of the reporter vector with the BAC-leading to the modified BAC-is facilitated by the presence of RecA. Recombinants are selected for by growth in the presence of chloramphenicol, ampicillin, and tetracycline. Only bacteria containing correctly modified BACs and copies of pSV1.RecA will be selected. Unmodified BACs (i.e., those lacking a pLD53.SC2/A-box insert) are eliminated by exposure to ampicillin. Free reporter plasmid remaining in the BAC host bacteria will also be eliminated, because this vector requires the π protein to replicate. The co-integrates are selected by growth at high temperature, thereby eliminating the RecA plasmid. Successful modification of the BAC-formation of the co-integrate-is confirmed in separate amplification reactions.