Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli

Abstract

Replacement of Escherichia coli's RecBCD function with phage lambda's Red function generates a strain whose chromosome recombines with short linear DNA fragments at a greatly elevated rate. The rate is at least 70-fold higher than that exhibited by a recBC sbcBC or recD strain. The value of the system is highlighted by gene replacement with a PCR-generated DNA fragment. The deltarecBCD::Plac-red kan replacement allele can be P1 transduced to other E. coli strains, making the hyper-Rec phenotype easily transferable.

FIG. 1

Linear DNA transformation substrate. Plasmid pKM131 contains the 2,557-bp PvuII lacZ fragment inserted into the PvuII site of pBR322, with a 1,100-bp kanamycin resistance-conferring fragment inserted into the BclI site within lacZ. When cut with BglI, pKM131 generates a fragment containing the kan gene, flanked by 921 and 1,200 bp of lacZ sequence. A similar plasmid, pKM133 (not shown), contains the tetracycline resistance determinant from pBR322, flanked by 1,256 and 1,301 bp of lacZ sequence.

FIG. 2

Linear transformation promoted by λ Red is IPTG dependent. Wild-type cells (W3110) containing λ Red-Gam-producing plasmid (pTP223) were incubated for various times in the presence of 1 mM IPTG prior to collection of the cells by centrifugation. Cells were made competent and transformed with equimolar amounts of lacZ::kan fragment (0.12 μg) and parent plasmid pKM131 (0.3 μg). Ratios of transformation titers (fragment/plasmid) were plotted as a function of time of exposure to IPTG.

FIG. 3

PCR analysis of lacZ::kan recombinants. PCRs were performed as described in Materials and Methods. Lanes: 1 and 7, 1-kb DNA ladder standards (Gibco-BRL); 2, PCR product from AB1157 containing pTP223; 3 to 5, PCR products from three independent linear transformants of AB1157 containing pTP223; 6, control reaction (no cells). The positions of 1- and 2-kb DNA standards are shown by arrows.

FIG. 4

Structures of recBCD deletions and P_lac-red substitution. Shown are the recBCD regions in wild-type cells (A), KM19 and KM21 (ΔrecBCD::kan) (B), and KM20 and KM22 (ΔrecBCD::P_lac-red kan) (C). Included are the relative positions of the PCR primers used to verify the structures of the wild-type and both substitution alleles. In panel C, transcription of red and kan occurs leftward (same direction as argA but opposite the direction of thyA).

FIG. 5

PCR analysis of the ΔrecBCD::P_lac-red allele. Lanes: 1, 1-kb DNA ladder standards (Gibco-BRL); 2 to 4, PCR products generated with primers 7, 8, and 9 (Fig. 4) from JC9387 (recBC sbcBC), KM20 (ΔrecBCD::P_lac-red sbcBC), and the control reaction (no cells), respectively; 5 to 7, PCR products generated with primers 10, 11, and 12 (Fig. 4) from JC9387 (recBC sbcBC), KM20 (ΔrecBCD::P_lac-red sbcBC), and the control reaction (no cells), respectively. The positions of 1- and 2-kb DNA standards are shown by arrows.

FIG. 6

UV resistance of cells containing the ΔrecBCD::P_lac-red allele. AB1157 (wild type; circles), KM21(ΔrecBCD; triangles), and KM22 (ΔrecBCD::P_lac-red; squares) were grown to 2 × 10⁸ cells/ml, spun down, resuspended in minimal medium, and kept on ice. Cells were diluted in minimal medium, spread on LB plates, exposed to the indicated doses of UV, and grown overnight in the dark. KM22 was grown and plated in the presence (closed squares) and absence (open squares) of 1 mM IPTG. Unirradiated plates were used to determine the total number of cells plated.