Fig. 4.
(A) Schematic representation of the two-step BAC modification system with a suicide plasmid. The Sp resistance marker (CrEcAadA) is shown as a dotted rectangle. (B) E. coli colony PCR using primers Hsp_AadA_BAC5 and AadA_BAC_3, indicating integration of CrEcAadA into the NAR2 gene of the resident BAC in 7 of 9 Cmr sucroser Aps colonies shown. Sp resistance correlated with the presence of CrEcAadA. Controls are the purified BAC 28J23 (B) and plasmid pALM30-Sp (P), shown next to the NEB 1-kb ladder. (C) Restriction digestion analysis of BAC 28J23 (WT) and the modified BAC 28J23-Sp (Mod), shown next to the NEB 1-kb ladder (*, 3-kb band). The differences, indicated by arrowheads, are as expected from the map of the modified plasmid, shown below. Note that the introduced cassette (black) contains new sites for EcoRV, MunI, PsiI, and SfiI.