A rapid seamless method for gene knockout in Pseudomonas aeruginosa
Abstract Background Pseudomonas aeruginosa is a model organism for the study of quorum sensing, biofilm formation, and also leading cause of nosocomial infections in immune compromised patients. As such P. aeruginosa is one of the most well studied organisms in terms of its genetics. However, the co...
Main Authors: | , |
---|---|
Format: | Article |
Language: | English |
Published: |
BMC
2017-09-01
|
Series: | BMC Microbiology |
Subjects: | |
Online Access: | http://link.springer.com/article/10.1186/s12866-017-1112-5 |
id |
doaj-10e495cfd5924cfb8aea603600a2106b |
---|---|
record_format |
Article |
spelling |
doaj-10e495cfd5924cfb8aea603600a2106b2020-11-24T21:02:16ZengBMCBMC Microbiology1471-21802017-09-011711810.1186/s12866-017-1112-5A rapid seamless method for gene knockout in Pseudomonas aeruginosaWeiliang HuangAngela Wilks0Department of Pharmaceutical Sciences, School of Pharmacy, University of MarylandAbstract Background Pseudomonas aeruginosa is a model organism for the study of quorum sensing, biofilm formation, and also leading cause of nosocomial infections in immune compromised patients. As such P. aeruginosa is one of the most well studied organisms in terms of its genetics. However, the construction of gene deletions and replacements in Pseudomonas aeruginosa is relatively time-consuming, requiring multiple steps including suicide vector construction, conjugation, inactivation with insertion of antibiotic resistance cassettes and allelic exchange. Even employing Gateway recombineering techniques with direct transformation requires a minimum two weeks. Methods We have developed a rapid streamlined method to create clean deletion mutants in P. aeruginosa through direct transformation, eliminating the need for the creation of Gateway-compatible suicide vectors. In this method, upstream and downstream sequences of the gene/locus to be deleted are amplified by polymerase chain reaction (PCR) and seamlessly fused with the linearized pEX18Tc sacB suicide plasmid by Gibson assembly. The resulting deletion plasmid is transformed into P. aeruginosa by an electroporation method optimized in this study. The plasmid is then integrated into the chromosome by homologous recombination, and deletion mutants are identified via sacB mediated sucrose counter-selection. Results The current method was employed to generate clean gene deletions of the heme assimilation system anti-σ factor, hasS and the virulence regulator involving ECF system anti-σ and σ factors vreA and vreI, respectively. The process from plasmid construction to confirmation by DNA sequencing of the gene deletion was completed in one week. Furthermore, the utility of the method is highlighted in the construction of the vreA and vreI deletions, where the start codon of vreA and the stop codon of vreI overlap. Utilizing Gibson assembly deletion mutants were constructed with single base pair precision to generate the respective vreA and vreI deletions, while maintaining the start and stop codon of the respective genes. Overall, this method allows for rapid construction of gene deletions in P. aeruginosa with base pair precision. Conclusion This method from the construction of the suicide vector to sequence confirmation of the unmarked gene deletion can be performed in one week, without the requirement for expensive proprietary reagents or instruments. The precision of Gibson assembly and the fact the accuracy in generating the desirable construct is 95%, makes this a viable and attractive alternative to previous methods.http://link.springer.com/article/10.1186/s12866-017-1112-5Pseudomonas AeruginosaGenetic knockoutGibson assembly |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Weiliang Huang Angela Wilks |
spellingShingle |
Weiliang Huang Angela Wilks A rapid seamless method for gene knockout in Pseudomonas aeruginosa BMC Microbiology Pseudomonas Aeruginosa Genetic knockout Gibson assembly |
author_facet |
Weiliang Huang Angela Wilks |
author_sort |
Weiliang Huang |
title |
A rapid seamless method for gene knockout in Pseudomonas aeruginosa |
title_short |
A rapid seamless method for gene knockout in Pseudomonas aeruginosa |
title_full |
A rapid seamless method for gene knockout in Pseudomonas aeruginosa |
title_fullStr |
A rapid seamless method for gene knockout in Pseudomonas aeruginosa |
title_full_unstemmed |
A rapid seamless method for gene knockout in Pseudomonas aeruginosa |
title_sort |
rapid seamless method for gene knockout in pseudomonas aeruginosa |
publisher |
BMC |
series |
BMC Microbiology |
issn |
1471-2180 |
publishDate |
2017-09-01 |
description |
Abstract Background Pseudomonas aeruginosa is a model organism for the study of quorum sensing, biofilm formation, and also leading cause of nosocomial infections in immune compromised patients. As such P. aeruginosa is one of the most well studied organisms in terms of its genetics. However, the construction of gene deletions and replacements in Pseudomonas aeruginosa is relatively time-consuming, requiring multiple steps including suicide vector construction, conjugation, inactivation with insertion of antibiotic resistance cassettes and allelic exchange. Even employing Gateway recombineering techniques with direct transformation requires a minimum two weeks. Methods We have developed a rapid streamlined method to create clean deletion mutants in P. aeruginosa through direct transformation, eliminating the need for the creation of Gateway-compatible suicide vectors. In this method, upstream and downstream sequences of the gene/locus to be deleted are amplified by polymerase chain reaction (PCR) and seamlessly fused with the linearized pEX18Tc sacB suicide plasmid by Gibson assembly. The resulting deletion plasmid is transformed into P. aeruginosa by an electroporation method optimized in this study. The plasmid is then integrated into the chromosome by homologous recombination, and deletion mutants are identified via sacB mediated sucrose counter-selection. Results The current method was employed to generate clean gene deletions of the heme assimilation system anti-σ factor, hasS and the virulence regulator involving ECF system anti-σ and σ factors vreA and vreI, respectively. The process from plasmid construction to confirmation by DNA sequencing of the gene deletion was completed in one week. Furthermore, the utility of the method is highlighted in the construction of the vreA and vreI deletions, where the start codon of vreA and the stop codon of vreI overlap. Utilizing Gibson assembly deletion mutants were constructed with single base pair precision to generate the respective vreA and vreI deletions, while maintaining the start and stop codon of the respective genes. Overall, this method allows for rapid construction of gene deletions in P. aeruginosa with base pair precision. Conclusion This method from the construction of the suicide vector to sequence confirmation of the unmarked gene deletion can be performed in one week, without the requirement for expensive proprietary reagents or instruments. The precision of Gibson assembly and the fact the accuracy in generating the desirable construct is 95%, makes this a viable and attractive alternative to previous methods. |
topic |
Pseudomonas Aeruginosa Genetic knockout Gibson assembly |
url |
http://link.springer.com/article/10.1186/s12866-017-1112-5 |
work_keys_str_mv |
AT weilianghuang arapidseamlessmethodforgeneknockoutinpseudomonasaeruginosa AT angelawilks arapidseamlessmethodforgeneknockoutinpseudomonasaeruginosa AT weilianghuang rapidseamlessmethodforgeneknockoutinpseudomonasaeruginosa AT angelawilks rapidseamlessmethodforgeneknockoutinpseudomonasaeruginosa |
_version_ |
1716775970045689856 |