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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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Schematic illustration of mutant fragment generation by overlap extension PCR. During first round PCR (PCR1), the 5' and 3' ends of the target genes, as well as the gentamycin (Gm) resistance cassette are amplified using four gene-specific primers (G-UpF-GWL, G-UpR-Gm, G-DnF-Gm and G-DnR-GWR) and the common Gm-specific primers (Gm-F and Gm-R). This generates three fragments with partial overlaps either to each other (indicated by the blue boxes signifying Gm overlap) or the attB1 and attB2 λ recombination sites (indicated by the green and pink boxes). These purified fragments are then assembled in vitro by overlap extension during second round PCR (PCR2) using common primers GW-attB1 and GW-attB2, resulting in a recombination-proficient mutant PCR fragment.
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Figure 2: Schematic illustration of mutant fragment generation by overlap extension PCR. During first round PCR (PCR1), the 5' and 3' ends of the target genes, as well as the gentamycin (Gm) resistance cassette are amplified using four gene-specific primers (G-UpF-GWL, G-UpR-Gm, G-DnF-Gm and G-DnR-GWR) and the common Gm-specific primers (Gm-F and Gm-R). This generates three fragments with partial overlaps either to each other (indicated by the blue boxes signifying Gm overlap) or the attB1 and attB2 λ recombination sites (indicated by the green and pink boxes). These purified fragments are then assembled in vitro by overlap extension during second round PCR (PCR2) using common primers GW-attB1 and GW-attB2, resulting in a recombination-proficient mutant PCR fragment.

Mentions: The basic strategy for generation of the mutant fragment by PCR overlap extension is illustrated in Fig. 2. In PCR1, the four gene-specific primers are used to amplify the 5' and 3' regions of the target gene. Simultaneously, two of the common primers, Gm-F and Gm-R, are used to amplify the Gmr cassette flanked by FRT sites from a pPS856 template. Since the Gmr fragment is used for all constructs, it can be prepared in larger amounts ahead of time and stored until needed. Fig. 3A illustrates typical results of PCR1 utilizing the example of P. aeruginosa gene PA1520, encoding a putative transcriptional regulator of the GntR family. When we adhered to the prescribed PCR conditions, the amplified fragments were very clean. It was especially prudent to follow the conditions for PCR amplification of the Gmr FRT fragment since the FRT sites possess significant secondary structures.


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

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

Schematic illustration of mutant fragment generation by overlap extension PCR. During first round PCR (PCR1), the 5' and 3' ends of the target genes, as well as the gentamycin (Gm) resistance cassette are amplified using four gene-specific primers (G-UpF-GWL, G-UpR-Gm, G-DnF-Gm and G-DnR-GWR) and the common Gm-specific primers (Gm-F and Gm-R). This generates three fragments with partial overlaps either to each other (indicated by the blue boxes signifying Gm overlap) or the attB1 and attB2 λ recombination sites (indicated by the green and pink boxes). These purified fragments are then assembled in vitro by overlap extension during second round PCR (PCR2) using common primers GW-attB1 and GW-attB2, resulting in a recombination-proficient mutant PCR fragment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1173109&req=5

Figure 2: Schematic illustration of mutant fragment generation by overlap extension PCR. During first round PCR (PCR1), the 5' and 3' ends of the target genes, as well as the gentamycin (Gm) resistance cassette are amplified using four gene-specific primers (G-UpF-GWL, G-UpR-Gm, G-DnF-Gm and G-DnR-GWR) and the common Gm-specific primers (Gm-F and Gm-R). This generates three fragments with partial overlaps either to each other (indicated by the blue boxes signifying Gm overlap) or the attB1 and attB2 λ recombination sites (indicated by the green and pink boxes). These purified fragments are then assembled in vitro by overlap extension during second round PCR (PCR2) using common primers GW-attB1 and GW-attB2, resulting in a recombination-proficient mutant PCR fragment.
Mentions: The basic strategy for generation of the mutant fragment by PCR overlap extension is illustrated in Fig. 2. In PCR1, the four gene-specific primers are used to amplify the 5' and 3' regions of the target gene. Simultaneously, two of the common primers, Gm-F and Gm-R, are used to amplify the Gmr cassette flanked by FRT sites from a pPS856 template. Since the Gmr fragment is used for all constructs, it can be prepared in larger amounts ahead of time and stored until needed. Fig. 3A illustrates typical results of PCR1 utilizing the example of P. aeruginosa gene PA1520, encoding a putative transcriptional regulator of the GntR family. When we adhered to the prescribed PCR conditions, the amplified fragments were very clean. It was especially prudent to follow the conditions for PCR amplification of the Gmr FRT fragment since the FRT sites possess significant secondary structures.

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