e36 Nucleic Acids Research, 2005, Vol. 33, No. 4 mutations like point mutations and clean deletions or introduce in-frame fusions of cDNAs or epitope tags without leaving at the same time a selectable marker or a loxP/Frt site at the modiﬁcation site. A handful of methods for making such muta- tions, all based on homologous recombination in E.coli, have been developed. One method is RecA dependent and relies on the use of a shuttle vector and two recombination steps: integ- ration followed by the resolution of the co-integrate (2,12). This method requires considerable up-front effort using tradi- tional cloning with restriction enzymes and ligation. A simpler and more widely used method is based on positive/negative selection using e.g. a sacB–neo fusion gene (8). neo (kana- mycin) resistance is used for positive selection while sucrose toxicity resulting from sacB expression is used for negative selection. A major drawback of this selection system is that spontaneous point mutations in the sacB portion of the sacB- neo fusion gene can occur without inﬂuencing the bacteria’s ability to grow on kanamycin. These sacB mutants signiﬁc- antly increase the background after negative selection. A related method is based on counterselection using a recogni- tion site for a rare restriction enzyme like I-SceI (13). For this method to work efﬁciently, the I-SceI restriction enzyme has to be induced in trans, and since there is no selection for maintaining the correct recognition sequence for this enzyme, the background due to point mutation or deletion of the restric- tion site is high. A method for BAC modiﬁcation without selection has also been developed (14). Although relatively efﬁcient, this method relies on a PCR-based screening of the resulting colonies to identify the desired clones. Since there is no selection step, the number of manipulations made possible by this selection procedure is more limited. Here, we report the development of a novel and highly efﬁcient galK-based positive/negative selection system for the manipulation of BACs. The E.coli galactose operon con- sists of four genes: galE, galT, galK and galM, which are necessary for growth and utilization of galactose as the only carbon source. The galK gene product, galactokinase, catalyzes the ﬁrst step in the galactose degradation pathway, phosphorylating galactose to galactose-1-phosphate. Galacto- kinase also efﬁciently catalyzes the phosphorylation of a galactose analog, 2-deoxy-galactose (DOG). The product of this reaction cannot be further metabolized, leading to a toxic build-up of 2-deoxy-galactose-1-phosphate (15). Thus, both positive and negative selection can be conferred by galK. Because galK is used for both selection steps, background following negative selection is reduced and no colony screening is necessary. The small size of the galK cassette (around 1200 bp plus homology arms) also makes it easier to amplify by PCR and to introduce into bacteria using electro- poration. MATERIALS AND METHODS General recombineering and galK selection Recombineering was performed as follows: 500 ml of an overnight culture was diluted in 25 ml Luria–Bertani (LB) medium with or without chloramphenicol selection (12.5 mg/ml) in a 50 ml bafﬂed conical ﬂask and grown at 32 C in a shaking waterbath to an OD600 of 0.6. Then, 10 ml was transferred to another bafﬂed 50 ml conical ﬂask and PAGE 2 OF 12 heat-shocked at 42 C for exactly 15 min in a shaking waterbath. The remaining culture was left at 32 C as the uninduced control. After 15 min the two samples, induced and uninduced, were brieﬂy cooled in an ice/waterbath slurry and then transferred to two 15 ml Falcon tubes (BD Biosciences) and pelleted using 5000 r.p.m. (eppendorf cen- trifuge 5804R, rotor A-4-44) at 0 C for 5 min. The supernatant was poured off and the pellet was resuspended in 1 ml ice-cold ddH2O by gently swirling the tubes in an ice/waterbath slurry. Subsequently, 9 ml ice-cold ddH2O was added and the samples pelleted again. This step was repeated once more. After the second washing and centrifugation step, the supernatant was removed, and the pellet ( 50 ml each) was kept on ice until electroporated with PCR product, gel-puriﬁed fragment or double-stranded oligo. An aliquot of 25 ml was used for each electroporation in a 0.1 cm cuvette (BioRad) at 25 mF, 1.75 kV and 200 V. After electroporation the bacteria were recovered in 1 ml LB (15 ml Falcon tube) for 1 h in a 32 C shaking waterbath. For the counterselection step (see below), the bacteria were recovered in 10 ml LB in a 50 ml bafﬂed conical ﬂask and incubated for 4.5 h in a 32 C shaking water- bath. After the recovery period, the bacteria were washed twice in 1· M9 salts as follows: 1 ml culture was pelleted in an eppendorf tube at 13 200 r.p.m. for 15 s and the super- natant was removed with a pipette. The pellet was resuspended in 1 ml of 1· M9 salts, and pelleted again. This washing step was repeated once more. After the second wash, the supernat- ant was removed and the pellet was resuspended in 1 ml of 1· M9 salts before plating serial dilutions (100 ml, 100 ml each of 1:10, 1:100 and 1:1000 dilutions) on minimal medium plates (see below). Washing in M9 salts is necessary to remove any rich media from the bacteria prior to selection on minimal medium. The uninduced samples routinely had a higher degree of lysis/bacterial death after electroporation, so the uninduced samples were diluted in 0.25–0.75 ml 1· M9 salts in the ﬁnal step to make up for the difference. Detailed protocols for recombineering can also be found at our website (http:// recombineering.ncifcrf.gov). Minimal media and indicator plates Minimal media and the indicator plates were prepared using standard methods (16). We added supplements as indicated. Washing solution: 1· M9 medium. Gal positive selection: M63 + agar (15 g/l; Difco, BD Biosciences) + D-galactose (0.2%; Sigma) + D-biotin (1 mg/l; Sigma) + L-leucine (45 mg/l; Sigma) and – chloramphenicol (12.5 mg/ml; Sigma). Gal counterselection: M63 + agar + glycerol (0.2%; Fischer) + D-biotin (1 mg/l) + L-leucine (45 mg/l) + DOG (0.2%; Ferro Pfanstiehl) and – chloramphenicol (12.5 mg/ml). Gal indicator plates: MacConkey agar (Difco, BD Biosciences) + D-galactose (1%) and – chloramphenicol (12.5 mg/ml). Plates were prepared using manufacturer’s instructions. Bacterial strains The strains used in this paper are listed in Table 1. SW101 was derived from DY380 by a homologous recombinational exchange of the mutated gal operon leader sequence with a