One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products

Abstract

We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.

Figure 1

A simple gene disruption strategy. H1 and H2 refer to the homology extensions or regions. P1 and P2 refer to priming sites.

Figure 2

Red recombinase expression plasmids. pKD20 and pKD46 (not shown) include 1,894 nt (31348–33241) and 2,154 nt (31088–33241) of phage λ (GenBank accession no. J02459), respectively.

Figure 3

Template plasmids. (A) Linear representations of the template plasmids. Arrowheads show locations and orientations of priming sites. P1: priming sites 0, 1, and 3; P2: priming site 2; P4: priming site 4. c1, c2, k1, k2, and kt: common test primers. (B) Sequences remaining after FLP-mediated excision of the antibiotic resistance genes. Priming sites 0, 1, and 3 begin at nucleotides 1, 2, and 3, respectively. Priming sites 2 and 4 begin at the left or right ends, as shown. Arrows with open arrowheads show the nearly perfect FRT site inverted repeats. The ribosome binding site (rbs) and methionine (met) start codon are marked.

Figure 4

Structures and PCR verification of DE(lacZYA)514 mutations. Top line shows the region near the wild-type lac operon. (A—C) Structures of the DE(lacZYA)514 alleles generated using pKD4 as template, pKD3 as template, and after elimination of the resistance genes, respectively. Numerals above the structures refer to locus-specific primers. The predicted PCR test products are shown. See Fig. 3 for other notations.

Figure 5

Structures of pstSCAB-phoU operon mutations. Top line shows the wild-type pstSCAB-phoU operon. Lower lines show the gene disruptions. The new junction fragments for the ΔpstS605, ΔpstCA607, and ΔpstB608 mutations were amplified by using primer 492 with 491, 497 with 498, and 495 with 496, respectively, and sequenced. See Fig. 4 for other notations.

Figure 6

Structures of other gene disruptions. See Fig. 4 for notations.