PAGE 5 OF 12 transferred to an eppendorf tube. An aliquot of 250 ml P2 buffer was added followed by mixing by inversion and incuba- tion for <5 min at room temperature. An aliquot of 250 ml N3 buffer was added followed by mixing and incubation on ice for 5 min. The supernatant was cleared by two rounds of centri- fugation at 13 200 r.p.m. for 5 min in a tabletop centrifuge. Each time the supernatant was transferred to a new tube. DNA was precipitated by adding 750 ml isopropanol, mixing and incubating on ice for 10 min, and centrifugation for 10 min at 13 200 r.p.m. The pellet was washed once in 70% ethanol and the dry pellet was dissolved in 50 ml TE. An aliquot of 40 ml ( 1 mg) was used for restriction analysis, and 1 ml was used as template for PCR analysis or transformation of electrocom- petent bacteria. Large-scale preparations of BAC DNA (25–100 mg) were done using the Nucleobond BAC maxi kit from Clontech (BD Biosciences) following the manufac- turer’s protocol. RESULTS Generation of SW102 cells To see whether we could develop a more efﬁcient selective system for BAC recombineering, we looked for a single select- able marker that could be used for both positive and negative selection. We focused on E.coli galK because both selection steps could be done using galK and its small size makes it easy to amplify by PCR. We previously reported the development of a bacterial strain, DY380 that is readily transformable with BAC DNA due to its DH10B origin (1). DY380 cells also harbor the defective l prophage required for recombineering. The defective prophage in DY380 was transferred into DH10B with a P1 phage lysate obtained from DY363 cells [for details, see (11)]. DY363 cells carry a 1200 bp IS2 insertion element, gal490, in the mRNA leader sequence of the galactose operon, preventing gal gene transcription. Since the galactose operon is directly proximal to the site of insertion of the defective Nucleic Acids Research, 2005, Vol. 33, No. 4 e36 prophage, the gal490 mutation was also transferred to DY380 during P1 transduction. Thus, DY380 is phenotypically Gal (galactose minus) and therefore unable to grow on galactose minimal medium. Recombineering was used to correct this problem. From the wild-type E.coli strain, W3110 (17), we PCR ampliﬁed a 441 bp fragment from the wild-type gal promoter, which spans the region containing the IS2 insertion element in DY380. This PCR product was introduced into DY380 and Gal+ recombin- ants were selected for growth on galactose minimal medium (see Materials and Methods). We named this strain SW101 (Table 1). This strain is identical to DY380 except that it lacks the IS2 insertion element and has been made wild type for the gal operon. We then made a precise deletion of the galK gene, leaving all other genes in the galactose operon intact. This was achieved by PCR amplifying two homology arms of 171 and 335 bp, respectively, ﬂanking the galK ORF and cloning them together into a plasmid using three-way ligation. Since the ORF of galK overlaps that of galM, we left behind the last 33 bp of galK (Figure 1) to allow for proper translation of galM. The targeting cassette, consisting of a 512 bp linear fragment, was electroporated into heat-induced and electro- competent SW101 cells, and the recombinant clones selected on minimal medium containing glycerol as the carbon source and DOG for selection against galK. The genotype of the resulting strain, SW102 (Table 1), was conﬁrmed by PCR analysis and sequencing of the modiﬁed region (Figure 1). This strain now harbors not only the defective l prophage, but also a functional gal operon, except for the deletion of galK. The l prophage is located between the galactose operon and the biotin operon, and the bio operon in these cells is nonfunctional, causing a biotin requirement. DH10B, and all derived strains, including SW102, are also deﬁcient in leucine metabolism (18). When using minimal medium, one therefore needs to add both biotin and leucine to the plates to allow SW102 growth (see Materials and Methods). Figure 1. Sequence analysis of the galactose operon in strains DY380 (A), SW101 (B) and SW102 (C). In SW102, the ORF of galK was deleted, leaving only 33 bp of galK behind to make sure that translation of galM is initiated properly. EcoRI: the restriction site used to clone the 50 and 30 homology arms flanking galK.