One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products
Kirill A. Datnko and Barry L. Wanner*
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907
Communicated by Jonathan Beckwith, Harvard Medical School, Boston, MA, April 11, 2000 (received for review February 13, 2000)
We have developed a simple and highly ef®cient method to disrupt chromosomal genes in Escherichia coli in which PCR primers pro-vide the homology to the targeted gene(s). In this procedure, recombination requires the phage l Red recombina, 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 ho
mologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are ¯anked 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, 演讲比赛技巧phnR, pstB, pstCAhigh five, 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 recombina which is also easily curable. This procedure should be widely uful, especially in genome analysis of E. coli and other bacteria becau the proce-dure can be done in wild-type cells.
bacterial genomics u FLP recombina u FRT sites u Red recombinachest
The availability of complete bacterial genome quences has provided a wealth of information on the molecular structure and organization of a myriad of genes and ORFs
who functions are poorly understood. A systematic mutational analysis of genes in their normal location can provide significant insight into their function. Although a number of general allele replacement methods (1±7) can be ud to inactivate bacterial chromosomal genes, the all require creating the gene disruption on a suitable plasmid before recombining it onto the chromosome. In con-trast, genes can be directly disrupted in Saccharomyces cerevisiae by transformation with PCR fragments encoding a lectable marker and having only 35 nt of flanking DNA homologous to the chromosome (8). This PCR-mediated gene replacement method has greatly facilitated the generation of specific mutants in the functional analysis of the yeast genome; it relies on the high efficiency of mitotic recombination in yeast (9). Directed disruption of chromosomal genes can also be done in 人教版六年级上册Candida albicans 劳动节 英文by using similar PCR fragments with 50- to 60-nt
homology extensions (10).
In contrast to yeast and a few naturally competent bacteria, most bacteria are not readily transformable with linear DNA. One reason Escherichia coli is not so transformable is bec
au of the prence of intracellular exonucleas that degrade linear DNA (11). However, recombination-proficient mutants lacking exonuclea V of the RecBCD recombination complex are transformable with linear DNA (12) . Recombination can occur in recB or recC mutants carrying a suppressor (sbcA or sbcB) mutation that activates an alternative recombination pathway; sbcA activates the RecET recombina of the Rac prophage, whereas 仰角sbcBprimary怎么读 enhances recombination by the RecF pathway (13). Such recBC sbcB mutants have been especially uful for recombining libraryin vitro constructed mutations onto the E. colidewen chromosome by using linear DNA (14). The discovery that recD
mutants are recombina proficient but lack exonuclea V (15, 16) has led to using singly mutated recDinsiston derivatives of E. coli (1) in similar gene disruption experiments.
It has been known for a long time that many bacteriophages encode their own homologous recombination systems (17). It has also recently been shown that the l Red (
g, b, exo ) function promotes a greatly enhanced rate of recombination over that exhibited by recBC sbcB or recD mutants when using linear DNA (18). Yet this system has produced no chromosomal gene disruptions when using PCR fragments with short homology extensions (unpublished data). A system has been developed that us the RecET recombina to disrupt plasmid-borne genes with such fragments (19); it has also been ud to make a single chromosomal deletion, but in that instance very long (138-nt) primers were ud.
Here we describe a procedure bad on the Red system that has allowed us to make more than 40 different disruptions on the E. coli chromosome without a single failure. The basic strategy is to replace a chromosomal quence (e.g., gene B in Fig. 1) with a lectable antibiotic resistance gene that is generated by PCR by using primers with 36 -nt homology extensions (H1 and H2). This is accomplished by Red-mediated recombination in the flanking homologies. After lection, the resistance gene can also be eliminated by using a helper plasmid expressing the FLP recombina, which acts on the directly repeated FRT (FLP recognition target) sites flanking the resistance gene. The
Red and FLP helper plasmids can be simply cured by growth at 37°C becau they are temperature-nsitive replicons.
