Table 1. Recombineering reagents used in this work PAGE 3 OF 12 Strains W3110 DH10B DY380 SW101 SW102 EL250 EL350 SW103 SW104 SW105 SW106 Selection cassette pgalK Genotype mcrA D(mrr-hsdRMS-mcrBC) DlacX74 deoR endA1 araD139 D(ara, leu) 7697 rpsL recA1 nupG f80dlacZDM15 galU galK DH10B [lc1857 (cro-bioA)<>Tet] galK+ gal490 DY380 gal+ SW101 DgalK DY380 (cro-bioA)<>araC-PBAD Flpe DY380 (cro-bioA)<>araC-PBAD Cre EL250 gal+ EL350 gal+ SW103 DgalK SW104 DgalK galK wild-type gene driven by the em 7 promoter Nucleic Acids Research, 2005, Vol. 33, No. 4 e36 Phenotype Gal+ Gal Gal Gal+ Gal Gal Gal Gal+ Gal+ Gal Gal Reference (17) (18) (11) This work This work (11) (11) This work This work This work This work This work <> indicates the result of a homologous recombination event. Gal+: the ability to use galactose as the sole carbon source. Gal : resistance to DOG and lack of the ability to grow on galactose as the sole carbon source. 441 bp PCR product (Expand High-Fidelity PCR System; Roche Applied Science) containing the wild-type gal operon leader sequence, made using W3110 (17) bacteria as the tem- plate. The primers used to amplify the wild-type leader were (Integrated DNA Technologies, Inc.): F 50-CGACGCATG- CAGGCATGAA-30 and R 50-AGTGGATCACGGTGTC- GATA-30 and the PCR conditions were: 94 C for 15 s, 60 C for 30 s and 72 C for 30 s, for 35 cycles. The PCR product was gel puriﬁed (GFX kit; Amersham Biosciences) and eluted in 50 ml ddH2O. An aliquot of 2.5 ml was used in the recombin- eering experiment as described above and the bacteria were plated on M63 minimal medium + galactose + leucine + biotin. The plates were incubated at 32 C for 2–3 days. A few of the many resulting colonies were streaked for single colonies on indicator plates and a single dark red Gal+ colony was used in the next step. SW102 was derived from SW101 in the following way: two homology arms ﬂanking the galK open reading frame (ORF) were PCR ampliﬁed using SW101 bac- teria as template and the following primers (Integrated DNA Technologies, Inc.); recognition sites for restriction enzymes are underlined: 50arm F: 50-AAATAACTCGAGCAGCTGC- ACGCGCACTTT-30; 50arm R: 50-AAATAAGAATTCTTCT- TACACTCCGGATTCGC-30; 30arm F: 50-AAATAAGAAT- TCTGTAAACCATCACAAGGAGCAG-30; 30arm R: 50-AA- TAAAGCGGCCGCCAGCTGGTTAACGCCCTGA-30. PCR conditions were 94 C for 15 s, 60 C for 30 s and 72 C for 30 s, for 35 cycles. The 50 homology arm (171 bp) was digested with XhoI and EcoRI, and the 30 arm (335 bp) with EcoRI and NotI. The digested PCR products were gel puriﬁed and triple- ligated into a vector digested with XhoI and NotI. The targeting cassette was then released from the backbone using XhoI and NotI digestion, followed by gel puriﬁcation. The cassette was eluted in 50 ml ddH2O, and 2.5 ml was used for recombineering as described above. After 4.5 h of outgrowth in LB media and two washes in 1· M9 salts, serial dilutions of the bacteria were plated on minimal plates containing glycerol as carbon source, leucine and biotin, plus DOG for selection against galK. The plates were incubated for 3 days at 32 C. A few of the many resulting colonies were streaked for single white/colorless (Gal ) colonies on indicator plates followed by PCR analysis and sequencing to check for correct deletion of the galK gene. A single, veriﬁed colony was expanded and a glycerol stock was made and used for initiation of all subsequent experiments. SW103 and SW104 were derived from EL250 and EL350, respectively, using the same method as for the derivation of SW101. SW105 and SW106 were derived from SW103 and SW104, respectively, using the same method as for the deriva- tion of SW102 from SW101. SW105 expresses Flp under the control of an arabinose-inducible promoter and SW106 expresses Cre under the same promoter. Construction of pgalK Two rounds of PCR were performed to construct a galK ORF driven by the prokaryotic em7 promoter. The ﬁrst round was performed using galK ORF 1st F and galK ORF R primers using W3110 bacteria (17) as template. The resulting PCR product was then used as template for a second round of PCR using galK ORF 2nd F and galK ORF R primers. The pro- duct from the second round of PCR was gel puriﬁed and digested with EcoRI and BamHI and cloned into EcoRI and BamHI digested pBluescript SK . pgalK was veriﬁed by sequencing [the plasmid sequence is available from our website (http://recombineering.ncifcrf.gov)].The primer sequences were (restriction sites and the galK ATG are under- lined and the em7 promoter is in italics): galK ORF 1st F: 50-CCCAGGAGGCAGATCATGAGTCTGAAAGAAAAAA- CTGAAAGAAAAAACACAATCTCTGT-30; galK ORF 2nd F: AATAAAGAATTCCTGTTGACAATTAATCATCG- GCATAGTATATCGGCATAGTATAATACGACAAGGTGA- GGAACTAAACCCAGGAGGCAGATCATG; galK ORF R: AATAAAGGATCCTCAGCACTGTCCTGCTCCTT-30. PCR conditions for both rounds were 94 C for 15 s, 60 C for 30 s and 72 C for 1.5 min, for 30 cycles. Primers were from Invitrogen. PCR amplification of the galK targeting cassette Sequences of all galK primers (Invitrogen) used in the experi- ments described in this paper are given below. em7-galK was PCR ampliﬁed using 1 ng pgalK as template and the following