Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange

Laura R Hmelo; Bradley R Borlee; Henrik Almblad; Michelle E Love; Trevor E Randall; Boo Shan Tseng; Chuyang Lin; Yasuhiko Irie; Kelly M Storek; Jaeun Jane Yang; Richard J Siehnel; P Lynne Howell; Pradeep K Singh; Tim Tolker-Nielsen; Matthew R Parsek; Herbert P Schweizer; Joe J Harrison

doi:10.1038/nprot.2015.115

Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange

Laura R Hmelo, Bradley R Borlee, Henrik Almblad, Michelle E Love, Trevor E Randall, Boo Shan Tseng, Chuyang Lin, Yasuhiko Irie, Kelly M Storek, Jaeun Jane Yang, Richard J Siehnel, P Lynne Howell, Pradeep K Singh, Tim Tolker-Nielsen, Matthew R Parsek, Herbert P Schweizer, Joe J Harrison

136 Citations (Scopus)

Abstract

Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knock-ins, as well as single-nucleotide insertions, deletions and substitutions, in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selections are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic-resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNANA. The entire procedure requires ~2 weeks.

Original language	English
Journal	Nature Protocols (Print)
Volume	10
Issue number	11
Pages (from-to)	1820-41
Number of pages	22
ISSN	1754-2189
DOIs	https://doi.org/10.1038/nprot.2015.115
Publication status	Published - 22 Oct 2015

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Hmelo, L. R., Borlee, B. R., Almblad, H., Love, M. E., Randall, T. E., Tseng, B. S., Lin, C., Irie, Y., Storek, K. M., Yang, J. J., Siehnel, R. J., Howell, P. L., Singh, P. K., Tolker-Nielsen, T., Parsek, M. R., Schweizer, H. P., & Harrison, J. J. (2015). Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nature Protocols (Print), 10(11), 1820-41. https://doi.org/10.1038/nprot.2015.115

Hmelo, LR, Borlee, BR, Almblad, H, Love, ME, Randall, TE, Tseng, BS, Lin, C, Irie, Y, Storek, KM, Yang, JJ, Siehnel, RJ, Howell, PL, Singh, PK, Tolker-Nielsen, T, Parsek, MR, Schweizer, HP & Harrison, JJ 2015, 'Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange', Nature Protocols (Print), vol. 10, no. 11, pp. 1820-41. https://doi.org/10.1038/nprot.2015.115

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title = "Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange",

abstract = "Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knock-ins, as well as single-nucleotide insertions, deletions and substitutions, in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selections are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic-resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNANA. The entire procedure requires ~2 weeks.",

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AB - Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knock-ins, as well as single-nucleotide insertions, deletions and substitutions, in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selections are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic-resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNANA. The entire procedure requires ~2 weeks.

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Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange

Abstract

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