An Efficient Recombination System for Chromosome Engineering in Escherichia Coli
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An efficient recombination system for chromosome engineering in Escherichia coli Daiguan Yu*, Hilary M. Ellis*, E-Chiang Lee†, Nancy A. Jenkins†, Neal G. Copeland†, and Donald L. Court*‡ *Gene Regulation and Chromosome Biology Laboratory and †Mouse Cancer Genetics Program, National Cancer Institute, Division of Basic Science, National Cancer Institute͞Frederick Cancer Research and Development Center, Frederick, MD 21702 Communicated by Allan Campbell, Stanford University, Stanford, CA, March 22, 2000 (received for review January 19, 2000) A recombination system has been developed for efficient chromo- frequencies of recombination with linear DNA containing long some engineering in Escherichia coli by using electroporated linear homologies by transforming in the presence of the bacteriophage DNA. A defective prophage supplies functions that protect and recombination functions (Exo and Beta) in a bacterial recBCD recombine an electroporated linear DNA substrate in the bacterial mutant background. This recombination system, like yeast, cell. The use of recombination eliminates the requirement for uses double strand break repair (10, 11). In a recBC sbcA strain, standard cloning as all novel joints are engineered by chemical short regions of homology can function in recombination of synthesis in vitro and the linear DNA is efficiently recombined into linear DNA; however, recombination is inefficient (12). In recϩ place in vivo. The technology and manipulations required are backgrounds, gene replacement on the chromosome has been simple and straightforward. A temperature-dependent repressor accomplished by integration and excision of episomes carrying tightly controls prophage expression, and, thus, recombination bacterial homology (13, 14). functions can be transiently supplied by shifting cultures to 42°C To obtain efficient recombination of linear donor DNA in for 15 min. The efficient prophage recombination system does not recAϩ or recAϪ backgrounds, an E. coli strain was made that require host RecA function and depends primarily on Exo, Beta, and contains a prophage harboring the recombination genes exo, Gam functions expressed from the defective prophage. The bet, and gam under control of a temperature-sensitive cI- defective prophage can be moved to other strains and can be easily repressor (Fig. 1). The exo, bet, and gam genes can be easily removed from any strain. Gene disruptions and modifications of switched on at 42°C and off at 32°C. When functions are turned both the bacterial chromosome and bacterial plasmids are possible. on for as short a time as 5 min, cells become more recombino- This system will be especially useful for the engineering of large genic and take up linear DNA without its destruction. Gam bacterial plasmids such as those from bacterial artificial chromo- inhibits the RecBCD nuclease from attacking linear DNA, and some libraries. Exo and Beta generate recombination activity for that linear DNA. More importantly, this recombination is proficient with NA engineering is conducted routinely in Escherichia coli, DNA homologies as short as 30–50 bp on the ends of linear DNA Dnot only for genetic studies in bacteria, but also for con- substrates. structing DNA molecules to be used in studies of other organ- isms. Most cloning methods use restriction endonuclease cleav- Materials and Methods age followed by DNA joining with DNA ligase. These in vitro Bacterial Strains. Bacterial strains used in this work are listed in cleavage and joining reactions are the basis for creating DNA Table 1. Strain DY329 was constructed by transduction of recombinants and for cloning of DNA segments on plasmid ZH1141 with P1 phage grown on WJW23 (see Table 1), selecting vectors in which additional modification and amplification can for nadA::Tn10 tetracycline resistance (TetR) at 32°C and then take place. In the yeast Saccharomyces cerevisiae, strategies that screening for the presence of a defective prophage, which generate novel DNA junctions exploit homologous recombina- causes temperature-sensitive cell growth at 42°C. Similar P1 tion (1). The DNA double-strand break and repair recombina- transduction was used to create other strains described in Table tion pathway is very efficient in yeast. Functions in this pathway 1 using standard media, methods, and selections (15). recombine transformed linear DNA with homologous DNA in the yeast cell. Moreover, this recombination is proficient with Induction of Recombination Functions and Preparation of Electro- short synthetic oligonucleotides, thereby permitting recombi- poration-Competent Cells. Overnight cultures grown at 32°C from nant DNA to be generated in vivo without using DNA restriction isolated colonies were diluted 50-fold in LB medium and were ϭ and ligation (2–4). However, subsequent manipulation of re- grown at 32°C with shaking to an OD600 0.4–0.6. Induction was combinant DNA molecules in yeast, as opposed to E. coli,is performed on a 10-ml culture in a baffled conical flask (50 ml) laborious, especially if it is to be used for recombinant DNA work by placing the flask in a water bath at 42°C with shaking (200 in other organisms. revolutions͞min) for 15 min. Immediately after the 15-min Unlike yeast, E. coli is not readily transformed by linear DNA induction, the flask was swirled in an ice water slurry to cool for fragments due in part to the rapid degradation of the DNA by 10 min. An uninduced control culture was also placed into the the intracellular RecBCD exonuclease (5). Mutant recBCD ice slurry. The cooled 10 ml cultures were centrifuged for 8 min strains lacking the exonuclease do not rapidly degrade linear at 5,500 ϫ g at 4°C. Each cell pellet was suspended in 1 ml of DNA; however, such strains are extremely poor growing, are ice-cold sterile water, was transferred to a 1.5-ml Eppendorf defective for recombination, and do not support efficient rep- lication of the many plasmids used in recombinant DNA work. In certain recAϩ backgrounds, the recBCD defect for recombi- Abbreviation: BAC, bacterial artificial chromosome. nation is suppressed, allowing linear DNA to be taken up by the ‡To whom reprint requests should be addressed at: Building 539͞Room 243, National ͞ cell and to be recombined (6). Other homologous recombination Cancer Institute, Division of Basic Science, National Cancer Institute Frederick Cancer Research and Development Center, Frederick, MD 21702. E-mail: [email protected]. strategies in E. coli recBC derivatives have been applied to ͞ The publication costs of this article were defrayed in part by page charge payment. This generate recombinants between homologies on linear and or article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. circular plasmids (7, 8). Still, recombinants are rare, the recom- §1734 solely to indicate this fact. bination generally uses thousands of base pairs of homology, and Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.100127597. plasmids are poorly maintained. Murphy (9) reported the highest Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.100127597 5978–5983 ͉ PNAS ͉ May 23, 2000 ͉ vol. 97 ͉ no. 11 Downloaded by guest on September 24, 2021 Fig. 1. Description of the defective prophage on the E. coli chromosome. The defective prophage contains genes from cI to int. A deletion (dotted line) removes the right side of the prophage from cro through attR and including bioA (21). On the chromosome, the nadA and gal operons are to the left of the prophage, and the bio genes without bioA are to the right. Genes of the prophage are shown on the solid line, and genes of the host are shown on the broken line. pL and pR indicate the early left and right promoters of . attL and attR indicate the left and right attachment sites of . The genes and functions have been described (17). tube, and was spun for 20 sec at 4°C at maximum speed in a the template DNA will generate the selected phenotype (i.e., the microfuge. After washing the cell pellets as described two more template is a plasmid), the template must be eliminated. Plasmid times, the cells were suspended in 100 l of ice-cold sterile water. template DNA can be destroyed by treatment with DpnI after This volume of competent cells is sufficient for two standard the PCR; DpnI cuts methylated GATC template DNA, leaving electroporation reactions (Ϸ108 cells per reaction). Larger cul- the newly replicated unmethylated DNA intact. Once a linear tures can be prepared for a greater number of reactions or for cassette has been generated, it can be stored and used as the Ϫ storage of electrocompetent cells at 80°C with 12% glycerol template for all subsequent PCRs. present. Fresh competent cells give highest efficiencies of re- combination and were used here. Cell Transformation. Purified linear donor DNA (1–10 l) was mixed with competent cells in a final volume of 50 l on ice and Preparation of Linear DNA Cassettes. Standard PCR conditions then was pipetted into a precooled electroporation cuvette (0.1 were used to amplify linear DNA fragments with the Expand cm). The amount of donor DNA used per reaction (usually 1–100 High Fidelity PCR system of Boehringer Mannheim. The chlor- R ng) is indicated for relevant experiments. Electroporation was amphenicol-resistant (Cm ) cassette cat was amplified from performed by using a Bio-Rad Gene Pulser set at 1.8 kV, 25 F pPCR-Script Cam (Stratagene) with primers 5ЈTGTGAC- Ј with Pulse controller of 200 ohms. Two protocols have been used GGAAGATCACTTCG and 5 ACCAGCAATAGACA- interchangeably to allow segregation of recombinant from pa- TAAGCG. The tetracycline-resistant (TetR) cassette tet was rental chromosomes within the electroporated cells. In both amplified from Tn10 with primers 5ЈCAAGAGGGTCAT- protocols, the electroporated cells were immediately diluted TATATTTCG and 5ЈACTCGACATCTTGGTTACCG. The with 1 ml of LB medium. In one, the cells were incubated for 1–2 ampicillin-resistant (ApR) cassette amp was amplified from pBluescript SK(ϩ) (Stratagene) with primers 5ЈCAT- h at 32°C before selecting for recombinants.