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An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants.

Choi KH, Schweizer HP - BMC Microbiol. (2005)

Bottom Line: Unmarked deletion mutants are finally obtained by Flp-mediated excision of the antibiotic resistance marker.The method was applied to deletion of 25 P. aeruginosa genes encoding transcriptional regulators of the GntR family.With appropriate modifications, the method should be applicable to other bacteria.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA.

ABSTRACT

Background: Traditional gene replacement procedures are still time-consuming. They usually necessitate cloning of the gene to be mutated, insertional inactivation of the gene with an antibiotic resistance cassette and exchange of the plasmid-borne mutant allele with the bacterial chromosome. PCR and recombinational technologies can be exploited to substantially accelerate virtually all steps involved in the gene replacement process.

Results: We describe a method for rapid generation of unmarked P. aeruginosa deletion mutants. Three partially overlapping DNA fragments are amplified and then spliced together in vitro by overlap extension PCR. The resulting DNA fragment is cloned in vitro into the Gateway vector pDONR221 and then recombined into the Gateway-compatible gene replacement vector pEX18ApGW. The plasmid-borne deletions are next transferred to the P. aeruginosa chromosome by homologous recombination. Unmarked deletion mutants are finally obtained by Flp-mediated excision of the antibiotic resistance marker. The method was applied to deletion of 25 P. aeruginosa genes encoding transcriptional regulators of the GntR family.

Conclusion: While maintaining the key features of traditional gene replacement procedures, for example, suicide delivery vectors, antibiotic resistance selection and sucrose counterselection, the method described here is considerably faster due to streamlining of some of the key steps involved in the process, especially plasmid-borne mutant allele construction and its transfer into the target host. With appropriate modifications, the method should be applicable to other bacteria.

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Gateway-recombinational cloning and return of the plasmid-borne deletion allele to the P. aeruginosa chromosome. The mutant DNA fragment generated by overlap extension PCR is first cloned into pDONR221 via the BP clonase reaction to create the entry clone pDONR221-Gene::Gm, which then serves as the substrate for LR clonase-mediated recombination into the destination vector pEX18ApGW. The resulting suicide vector pEX18ApGW-Gene::Gm is then transferred to P. aeruginosa and the plasmid-borne deletion mutation is exchanged with the chromosome to generate the desired deletion mutant. Please note that, as discussed in the text, gene replacement by double-crossover can occur quite frequently, but it can also be a rare event in which case allele exchange happens in two steps involving homologous recombination. First, the suicide plasmid is integrated via a single-crossover event resulting in generation of a merodiploid containing the wild-type and mutant allele. Second, the merodiploid state is resolved by sacB-mediated sucrose counterselection in the presence of gentamycin, resulting in generation of the illustrated chromosomal deletion mutant. An unmarked mutant is then obtained after Flp recombinase-mediated excision of the Gm marker.
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Figure 4: Gateway-recombinational cloning and return of the plasmid-borne deletion allele to the P. aeruginosa chromosome. The mutant DNA fragment generated by overlap extension PCR is first cloned into pDONR221 via the BP clonase reaction to create the entry clone pDONR221-Gene::Gm, which then serves as the substrate for LR clonase-mediated recombination into the destination vector pEX18ApGW. The resulting suicide vector pEX18ApGW-Gene::Gm is then transferred to P. aeruginosa and the plasmid-borne deletion mutation is exchanged with the chromosome to generate the desired deletion mutant. Please note that, as discussed in the text, gene replacement by double-crossover can occur quite frequently, but it can also be a rare event in which case allele exchange happens in two steps involving homologous recombination. First, the suicide plasmid is integrated via a single-crossover event resulting in generation of a merodiploid containing the wild-type and mutant allele. Second, the merodiploid state is resolved by sacB-mediated sucrose counterselection in the presence of gentamycin, resulting in generation of the illustrated chromosomal deletion mutant. An unmarked mutant is then obtained after Flp recombinase-mediated excision of the Gm marker.

Mentions: The BP and LR clonase reactions (Fig. 4) were performed according to Invitrogen's (Carlsbad, CA) Gateway cloning manual. For the BP clonase reaction pDONR221 was used, but only half of the recommended BP clonase enzyme mix. Gmr and kanamycin resistant (Kmr) DH5α or HPS1 transformants were selected and the presence of the correct plasmids was confirmed by XbaI digestion (the FRT sites flanking the Gmr gene each contain a single XbaI site). The insert of a correct pDONR-Gene::Gm entry clone was then recombined into the destination vector pEX18ApGW using the LR clonase protocol, again using only half of the recommended amount of LR clonase mix. Recombinants were transformed into DH5α and Apr colonies were selected. We noticed that, at least under the conditions we used, many transformants exhibited a Apr Kmr phenotype, indicating that they either contained both pDONR-Gene::Gm and pEX18ApGW-Gene::Gm or, more likely, a cointegrate of both plasmids. The presence of the correct pEX18ApGW-Gene::Gm in transformants that were only Apr was confirmed by XbaI digestion.


An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants.

Choi KH, Schweizer HP - BMC Microbiol. (2005)

Gateway-recombinational cloning and return of the plasmid-borne deletion allele to the P. aeruginosa chromosome. The mutant DNA fragment generated by overlap extension PCR is first cloned into pDONR221 via the BP clonase reaction to create the entry clone pDONR221-Gene::Gm, which then serves as the substrate for LR clonase-mediated recombination into the destination vector pEX18ApGW. The resulting suicide vector pEX18ApGW-Gene::Gm is then transferred to P. aeruginosa and the plasmid-borne deletion mutation is exchanged with the chromosome to generate the desired deletion mutant. Please note that, as discussed in the text, gene replacement by double-crossover can occur quite frequently, but it can also be a rare event in which case allele exchange happens in two steps involving homologous recombination. First, the suicide plasmid is integrated via a single-crossover event resulting in generation of a merodiploid containing the wild-type and mutant allele. Second, the merodiploid state is resolved by sacB-mediated sucrose counterselection in the presence of gentamycin, resulting in generation of the illustrated chromosomal deletion mutant. An unmarked mutant is then obtained after Flp recombinase-mediated excision of the Gm marker.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1173109&req=5

Figure 4: Gateway-recombinational cloning and return of the plasmid-borne deletion allele to the P. aeruginosa chromosome. The mutant DNA fragment generated by overlap extension PCR is first cloned into pDONR221 via the BP clonase reaction to create the entry clone pDONR221-Gene::Gm, which then serves as the substrate for LR clonase-mediated recombination into the destination vector pEX18ApGW. The resulting suicide vector pEX18ApGW-Gene::Gm is then transferred to P. aeruginosa and the plasmid-borne deletion mutation is exchanged with the chromosome to generate the desired deletion mutant. Please note that, as discussed in the text, gene replacement by double-crossover can occur quite frequently, but it can also be a rare event in which case allele exchange happens in two steps involving homologous recombination. First, the suicide plasmid is integrated via a single-crossover event resulting in generation of a merodiploid containing the wild-type and mutant allele. Second, the merodiploid state is resolved by sacB-mediated sucrose counterselection in the presence of gentamycin, resulting in generation of the illustrated chromosomal deletion mutant. An unmarked mutant is then obtained after Flp recombinase-mediated excision of the Gm marker.
Mentions: The BP and LR clonase reactions (Fig. 4) were performed according to Invitrogen's (Carlsbad, CA) Gateway cloning manual. For the BP clonase reaction pDONR221 was used, but only half of the recommended BP clonase enzyme mix. Gmr and kanamycin resistant (Kmr) DH5α or HPS1 transformants were selected and the presence of the correct plasmids was confirmed by XbaI digestion (the FRT sites flanking the Gmr gene each contain a single XbaI site). The insert of a correct pDONR-Gene::Gm entry clone was then recombined into the destination vector pEX18ApGW using the LR clonase protocol, again using only half of the recommended amount of LR clonase mix. Recombinants were transformed into DH5α and Apr colonies were selected. We noticed that, at least under the conditions we used, many transformants exhibited a Apr Kmr phenotype, indicating that they either contained both pDONR-Gene::Gm and pEX18ApGW-Gene::Gm or, more likely, a cointegrate of both plasmids. The presence of the correct pEX18ApGW-Gene::Gm in transformants that were only Apr was confirmed by XbaI digestion.

Bottom Line: Unmarked deletion mutants are finally obtained by Flp-mediated excision of the antibiotic resistance marker.The method was applied to deletion of 25 P. aeruginosa genes encoding transcriptional regulators of the GntR family.With appropriate modifications, the method should be applicable to other bacteria.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA.

ABSTRACT

Background: Traditional gene replacement procedures are still time-consuming. They usually necessitate cloning of the gene to be mutated, insertional inactivation of the gene with an antibiotic resistance cassette and exchange of the plasmid-borne mutant allele with the bacterial chromosome. PCR and recombinational technologies can be exploited to substantially accelerate virtually all steps involved in the gene replacement process.

Results: We describe a method for rapid generation of unmarked P. aeruginosa deletion mutants. Three partially overlapping DNA fragments are amplified and then spliced together in vitro by overlap extension PCR. The resulting DNA fragment is cloned in vitro into the Gateway vector pDONR221 and then recombined into the Gateway-compatible gene replacement vector pEX18ApGW. The plasmid-borne deletions are next transferred to the P. aeruginosa chromosome by homologous recombination. Unmarked deletion mutants are finally obtained by Flp-mediated excision of the antibiotic resistance marker. The method was applied to deletion of 25 P. aeruginosa genes encoding transcriptional regulators of the GntR family.

Conclusion: While maintaining the key features of traditional gene replacement procedures, for example, suicide delivery vectors, antibiotic resistance selection and sucrose counterselection, the method described here is considerably faster due to streamlining of some of the key steps involved in the process, especially plasmid-borne mutant allele construction and its transfer into the target host. With appropriate modifications, the method should be applicable to other bacteria.

Show MeSH
Related in: MedlinePlus