JOURNAL OF BACTERIOLOGY, July 1993, p. 4260-4262 Vol. 175, No. 13 0021-9193/93/134260-03$02.00/0 Copyright © 1993, American Society for Microbiology Simple Phagemid-Based System for Generating Allele Replacements in Escherichia coli STEVEN SLATERt AND RUSSELL MAURER* Department ofMolecular Biology and Microbiology, School ofMedicine, Case Western Reserve University, Cleveland, Ohio 44106-4960

Received 5 March 1993/Accepted 30 April 1993

We describe a phagemid-based system for rapidly generating chromosomal allele replacements in two steps. The system utilizes simple vector-determined selection procedures for each step and requires only that the target strain be male and sup' (nonsuppressing for amber mutations).

Dotto and Horiuchi (2) observed that when two phage fl at 600 nm (OD6.) of approximately 0.2. Helper phage replication origins are present on a single in identical f1R189 (obtained from George Weinstock) was then added at orientations, infection of the host by fl helper phage a multiplicity of infection of approximately 20. After the leads to initiation of plus strand synthesis at one origin and culture was grown overnight, phages and phagemids were termination at the second origin. This process produces and separated from the cells by centrifugation and filtration packages a single-stranded, circular progeny molecule in through a 0.45-,um-pore-size membrane. Phage f1R189, a which DNA downstream of the second origin is deleted (Fig. II amber mutant, was used as helper because it will not 1). Accordingly, a plasmid having its ColEl origin within the replicate in the sup+ target strain. Since an intact M13 origin region to be deleted would, upon processing by helper remains on the deleted plasmid, and a molecule bearing an phage, generate a molecule incapable of autonomous repli- M13 origin will replicate in the presence of gene II protein, cation. We demonstrate here the use of such a plasmid for use of a gene II amber mutant reduced the number of Kanr chromosomal allele replacement in Escherichia coli. colonies arising from coinfection of a sup+ cell with helper A generalized map of plasmid pBIP (bacterial integrating phage and a deleted pBIP. plasmid) is shown at the top of Fig. 1. This plasmid, which Serial 10-fold dilutions of the pFF579/f1R189 lysate were was derived from pZ150 (6), contains an ntp-sacB cartridge, made, and 100 ,ul of each dilution was mixed separately with allowing both positive (kanamycin) and negative (5% su- 5 ml of late-log-phase recipient cells (RM1094; HfrH lac+ crose) selection of the vector (4). It also carries an intact sup+). The mixture was incubated with aeration at 37°C for M13 replication origin, a polylinker, and a second region of 1 h to allow expression of antibiotic M13 DNA, designated M13 ter. M13 ter is the 212-bp resistance, and then NaeI-AvaI fragment containing region A (3) of the plus each culture was concentrated by centrifugation, resus- strand origin from M13mpl8 (5). Region A has pended in 0.1 ml of LB broth (containing [per liter] 10 g of all sequences necessary for termination of DNA replication tryptone, 5 g of yeast extract, 5 g of NaCl, and 1 ml of 1 N but lacks certain initiation NaOH), and plated on LB plates containing kanamycin. sequences. Thus, single-stranded The pFF579/f1R189 lysate contained Kanr transducing par- DNA synthesis originates only from the intact M13 origin ticles at a proximal to npt. Synthesis proceeds through npt, sac, and frequency of 6 x 10-6 relative to the number of the DNA viable phage (assayed in a supE strain). Much of this polylinker (and any cloned therein) and terminates difference may be attributed to the fact that phagemid DNA at M13 ter. The plasmid ColEl origin is never replicated must during plus strand synthesis, so the final product is a integrate to yield a Kanr transductant. An undiluted covalently closed, single-stranded DNA molecule lacking lysate of pBIP itself (i.e., lacking E. coli homology) pro- the ColEl origin. The deleted molecule, although incapable duced a single Kanr colony, for a frequency of approxi- of autonomous replication, can be maintained by integration mately 10-' relative to the number of helper phage. This into the E. coli . colony was not characterized. In the experiments for which the results are shown in Fig. Deletion of the pBIP ColEl origin following fl-mediated 2 and 3, we show the integration of the ColEl-deleted replication is expected to reduce the size of a HindIII-PstI plasmid via lacZ homology. Moreover, using a marked lacZ oni-containing fragment from 3.1 to 0.9 kb. This deletion is gene, we show that subsequent plasmid excision can lead to clearly shown by Southern blot analysis of the pFF579 origin chromosomal allele replacement. Plasmid pFF579 is a pBIP region in RM4048, a strain with pFF579 integrated at lacZ derivative that carries lacZ with a 624-bp HpaI fragment (Fig. 2). Figure 3, lane 2, shows that the integration occurred internal to lacZ replaced by a 1.3-kb fragment carrying cat by a crossover in the region of homology 3' of the disruption (chloramphenicol resistance). The Camr marker is flanked 5' in lacZ. and 3' by 450 and 2,000 bp, respectively, of lacZ DNA. E. To complete the allele exchange, five colonies of strain coli JM109 (F+ supE recA) harboring pFF579 was grown in RM4048 were grown separately to the late log phase in LB 2x YT broth (3a) containing kanamycin to an optical density broth. Each culture was diluted serially, a 100-,ul aliquot of each dilution was plated on salt-free LB agar containing 5% sucrose, and the plates were incubated overnight at 30°C (1). * Corresponding author. Electronic mail address: RAM3@po. Sucr colonies were checked for kanamycin sensitivity, and cwru.edu. the physical structure of the lac region of several Sucr Kans t Present address: Department of Biochemistry and Molecular strains was assayed by Southern blot. Figure 3, lanes 3 and Biology, Harvard University, Cambridge, MA 02138-2019. 4, demonstrates both allele replacement and restoration of 4260 VOL. 175, 1993 NOTES 4261

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PROBE FIG. 3. (A) Southern blot analysis of the lacZ locus. Lanes: 1, RM1094 (lacZ'); 2, RM4048 (pFF579 integrated at lacZ); 3, RM4049 (lacZ::cat derivative of RM4048); 4, RM4050 (lacZ' derivative of RM4048). - All samples were digested with BssHII and probed with the a-complementing fragment of lacZ from pUC19 (5). Positions and sizes (in kilobase pairs) of marker DNA molecules are shown to the left. (B) Structures of lacZ' (top), pFF579 integrated at lacZ (middle), and lacZ::cat (bottom). The letter B designates a BssHII site.

FIG. 1. Structure of pBIP and a pBIP derivative produced by M13- or fl-mediated replication. Plus strand synthesis initiates at the intact the parental allele in different resolution products derived M13 origin ofpBIP, proceeds through npt, sac, and the polylinker, and then terminates at M13 ter. A covalently closed, single-stranded DNA from RM4048. As expected, resolution resulted in loss of lacking the ColEl origin results (bottom). Plasmid pBIP3 (7.7 kb), the vector sequences (Fig. 2, lanes 3 and 4). In these experi- most useful pBIP construct, carries the pBluescript II SK+ polylinker ments, 1 to 3% of the Sucr colonies analyzed were Lac (Stratagene) and has unique ScaI, ApaI, SpeI, NotI, BstXI, and SacI mutants (Table 1). This suggests a disproportionate bias, at sites. The SacI site is proximal to M13 ter. least in this case, for resolution in the longer homology region. Selection on sucrose plates containing chloramphen- icol yielded 100% Lac mutants. Two Lac' Sucr mutants (probably siblings) in culture 1 were also Cams but Kanr. Although the reason for this phenotype was not determined, A 1 2 3 4 the result is consistent with a deletion from sacB through 5- cat. I These data demonstrate the feasibility of chromosomal 3- allele exchange by using the pBIP system. We and others 2- 1 have used this system for exchange of both point mutations 9_ and large insertions in at least five additional . The 1- protocol is rapid and completely general, imposing minimal requirements on the target strain. The procedure incorpo- 0.5 -

TABLE 1. Frequency of lacZ mutants induced by resolution of B the integrated pFF579 in strain RM4048 M13 ter ColEl ori Ml 3 ori I' Distribution of Kan' Sucr Pst Culture Sucr colonies coloniesb (no. of colonies) Hindlil no. () Ml 3 ori Lac- Camr Lac' Cams 1 1 KB 1 4 1 97 HindlIn I11 Psitl 2 6 3 97 FIG. 2. (A) Southern blot analysis of the pBIP origin region. 3 4 3 97 Lanes: 1, pBIP; 2, RM4048 (pFF579 integrated at lacZ); 3, RM4049 4 0.4 1 99 (lacZ::cat derivative of RM4048); 4, RM4050 (lacZ+ derivative of 5 6 1 99 RM4048). All samples were digested with PstI and HindIII and a with the PstI-HindIII Posi- In experiments in which genes other than lacZ were disrupted, we found probed pBIP oni-containing fragment. the number of Sucr colonies in a culture to be as few as 1 in 106 (data not tions and sizes (in kilobase pairs) of marker DNA molecules are shown). shown to the left. (B) Structure of the pBIP origin region (top) and b For each culture, 100 Sucr colonies were scored. Culture 1 produced two the pBIP origin region following fl-mediated replication (bottom). Lac' Kanr Cams colonies (see the text). 4262 NOTES J. BACTERIOL.

rates strong selection for both the integration and excision sacB gene and a temperature-sensitive pSC101 . Mol. steps and is suitable for exchanging phenotypically silent Microbiol. 5:1447-1457. alleles. Since no plasmid or transposon sequences are re- 2. Dotto, G. P., and K. Horiuchi. 1981. Replication of a plasmid tained, the same process can be used sequentially to effect containing two origins of bacteriophage fl. J. Mol. Biol. 153:169- complex strain constructions without reliance on the numer- 176. 3. Dotto, G. P., K. Horiuchi, and N. D. Zinder. 1982. The functional ous (and potentially limiting) antibiotic-resistance markers origin of bacteriophage fl DNA replication. Its signals and used conventionally. domains. J. Mol. Biol. 172:507-521. 3a.Miller, J. H. 1972. Experiments in molecular genetics, p. 433. We thank Peter Berget for discussions of M13 biology leading to Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. production of pBIP, George Weinstock for f1R189, Alan Collmer for 4. Ried, J. L., and A. Collmer. 1987. An nptl-sacB-sacR cartridge the sac-npt cassette, and Miriam Lifsics and Craig Altier for helpful for constructing directed, unmarked mutations in gram-negative discussions. by marker exchange-eviction mutagenesis. Gene 57:239- This work was supported by Public Health Service grants 246. GM39419 and GM47111 from the National Institutes of Health. 5. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage vectors and host strains: se- REFERENCES quences of the M13mpl8 and pUC19 vectors. Gene 33:103-119. 6. Zagursky, R. J., and M. L. Berman. 1984. Cloning vectors that 1. Blomfield, I. C., V. Vaughn, R. F. Rest, and B. L Eisenstein. 1991. yield high levels of single-stranded DNA for rapid DNA sequenc- Allelic exchange in Eschenchia coli using the Bacillus subtilis ing. Gene 27:183-191.