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

TY - JOUR

T1 - Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange

AU - Hmelo, Laura R

AU - Borlee, Bradley R

AU - Almblad, Henrik

AU - Love, Michelle E

AU - Randall, Trevor E

AU - Tseng, Boo Shan

AU - Lin, Chuyang

AU - Irie, Yasuhiko

AU - Storek, Kelly M

AU - Yang, Jaeun Jane

AU - Siehnel, Richard J

AU - Howell, P Lynne

AU - Singh, Pradeep K

AU - Tolker-Nielsen, Tim

AU - Parsek, Matthew R

AU - Schweizer, Herbert P

AU - Harrison, Joe J

PY - 2015/10/22

Y1 - 2015/10/22

N2 - 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.

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.

U2 - 10.1038/nprot.2015.115

DO - 10.1038/nprot.2015.115

M3 - Journal article

C2 - 26492139

SN - 1754-2189

VL - 10

SP - 1820

EP - 1841

JO - Nature Protocols (Print)

JF - Nature Protocols (Print)

IS - 11

ER -