e36 Nucleic Acids Research, 2005, Vol. 33, No. 4 galK expression cassette Having produced a bacterial strain, SW102, which is galK defective, the next step was to make a galK expression cassette that could be used to restore the bacteria’s ability to grow on galactose by providing galK in trans. This was achieved by PCR ampliﬁcation of the wild-type galK ORF from W3110 cells. We then added a minimal bacterial promoter, em7, using a two-step PCR approach (see Materials and Methods), and cloned the expression cassette into pBluescript SK . We call this plasmid pgalK (Table 1). The constitutively active galK expression cassette can easily be ampliﬁed by PCR with homology arms added to the primers (see Materials and Methods). Making a single base pair substitution The general scheme for making mutations in BACs using galK selection is depicted in Figure 2. To test the galK selection system for BAC recombineering, we decided to introduce a point mutation into a BAC containing the murine Nras gene (CITB clone 50J2). The sequence of the glycine-coding codon 12 is GGT. By changing this codon to GAT we would obtain the desired mutation, G12D. In order to introduce this muta- tion into the BAC, we ﬁrst ampliﬁed the galK expression cassette by PCR using primers with 50 bp of homology to either side of the second position of the GGT codon. Following homologous recombination, this targeting would introduce a 1 bp deletion into codon 12 in addition to inserting the galK selection cassette. Instead of deleting the basepair, the galK cassette could have been inserted right next to the basepair instead. SW102 cells containing the 50J2 BAC were heat- induced and made electrocompetent, and then electroporated with the galK cassette. Gal+ recombinant colonies were selec- ted for growth on galactose minimal medium with chloram- phenicol to maintain the BAC. Bacteria grow more slowly on minimal media than on rich media, and we generally pick colonies after 2–3 days. For this ﬁrst step, we do not expect any background colonies on the non-induced control plates if the pgalK plasmid is properly eliminated (see Materials and Methods). To purify the Gal+ colonies, we streaked a few colonies on MacConkey galactose indicator plates to obtain single, bright pink/red Gal+ colonies. One of these single- cloned colonies was picked to initiate a culture for the next step, counterselection. We ﬁnd that there is no need to analyze the Gal+ colonies further, before proceeding to counterselection. A 100 bp dsDNA oligo was then prepared by annealing two complementary oligos having 49 and 50 bp homology, respectively, to either side of the desired mutation, a single A/T bp. An aliquot of 200 ng of this oligo was then electro- porated into heat-induced and electrocompetent SW102 Gal+ cells containing the galK modiﬁed 50J2 BAC. After electro- poration, the bacteria were allowed 4.5 h outgrowth in a 32 C shaking waterbath. The bacteria were then washed in M9 salts to remove any rich medium, and plated on minimal medium with glycerol, DOG and chloramphenicol. The 4.5 h out- growth is necessary to obtain complete segregation of the recombinant BACs containing the mutation. After 3 days, we obtained colonies with a ratio of 10–100:1 when comparing plates with heat-induced to non-induced bacteria. We picked 12 colonies from the heat-induced plates for BAC minipreps, PAGE 6 OF 12 Figure 2. Overview of the galK selection scheme. The result of the first targeting event is the insertion of constitutively active galK into a defined position on the BAC by selection on minimal medium containing galactose and chloramphenicol to select for the maintenance of the BAC. The bacteria are now phenotypically Gal+. Next, the galK cassette is replaced by a dsDNA oligo, a PCR product, or a cloned dsDNA fragment carrying a desired mutation (indicated by a star) and flanked by the same homology arms used in the first selection step. This is achieved by negative selection using minimal medium containing 2-deoxy-galactose (DOG) with glycerol as the sole carbon source. The bacteria become phenotypically Gal . H1 and H2, homology arms 1 and 2, respectively; cat, chloramphenicol acetyl transferase gene; ori2, BAC origin of replication; galK, E.coli galactokinase gene driven by a minimal promoter. followed by SpeI restriction analysis (Figure 3A). Ten out of the twelve clones had the same restriction pattern as the unmodiﬁed 50J2 BAC DNA, suggesting that the desired replacement of the galK gene by the point mutation had occurred. This was conﬁrmed by PCR ampliﬁcation followed