Table 2 Plasmids

pKM208

[34]. bla-Ptac-gam-bet-exo-lacI, temperature-sensitive mutant pSC101 origin

pJL148

[16]. SPA-neo

pTP1068

Plasmid containing bla-P₃₂-aacC. See the description of pTP1232 below.

pTP1205

pKM208 × SP01,02 pcr of pJL148. The Red-mediated recombination event fuses the SPA tag to the C-terminal end of bet, deletes most of exo, and adds neo.

pTP1206

Deletion of bla from pTP1205 by digestion with PvuI and religation

pTP1214

pKM208 × cat29,30 pcr of Tn9 cat. The Red-mediated recombination event replaces a segment of pKM208 between lacI and the replication origin with the cat gene, flanked by BamH1 sites.

pTP1215

Deletion of cat from pTP1214 by digestion with BamH1 and religation

pTP1216

pTP1215 × cat27,28 pcr of Tn9 cat. The Red-mediated recombination event replaces bla and the f1 replication origin with cat. Smaller, chloramphenicol resistance-conferring version of pKM208.

pTP1222

A segment of λ wild type DNA was amplified by SR2,3 pcr. The product was digested with BamH1 and XbaI, and ligated with a BamH1- and XbaI-ended plasmid segment consisting of the Tn903 aph gene and the pSC101 replication origin.

pTP1223

Deletion of sequences between the EcoRV and XbaI sites of pTP1222. The resulting plasmid contains λ sequences from 45262 in the S gene to 45828 in the R gene.

pTP1228, 1230, 1231

Wild type, D41N, and E44K alleles of greA, respectively, amplified by PCR with primers greA5 and greA6, and cloned between the EcoR1 and BamH1 sites of a derivative of pBR322 lacking the sequences between the BamH1 and PvuII sites.

pTP1232

Contains the sequences used to construct TP1234, DNA from which in turn was used as the template for PCR synthesis of the 587-bp dsDNA used in the tests of Red-mediated chromosomal gene replacement (sequence shown in Figure 4). It includes the bla gene of pBR322, the synthetic, unregulated promoter P₃₂ (closely related to CP32 [46]), the lacZ ribosome binding site, and the N-terminal end of lacZ.