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

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 similar to 2 weeks.

Borlee, Bradley R. (author)
Almblad, Henrik (author)
Love, Michelle E. (author)
Randall, Trevor E. (author)
Tseng, Boo Shan (author)
Lin, Chuyang (author)
Irie, YasuhikoUmeå universitet,Institutionen för molekylärbiologi (Medicinska fakulteten)(Swepub:umu)yair0002 (author)
Storek, Kelly M. (author)
Yang, Jaeun Jane (author)
Siehnel, Richard J. (author)
Howell, P. Lynne (author)
Singh, Pradeep K. (author)
Tolker-Nielsen, Tim (author)
Parsek, Matthew R. (author)
Schweizer, Herbert P. (author)
Harrison, Joe J. (author)
Umeå universitetInstitutionen för molekylärbiologi (Medicinska fakulteten) (creator_code:org_t)

In:Nature Protocols: Springer Science and Business Media LLC10:11, s. 1820-18411754-21891750-2799

By the author/editor: Hmelo, Laura R.; Borlee, Bradley ...; Almblad, Henrik; Love, Michelle E ...; Randall, Trevor ...; Tseng, Boo Shan; show more...; Lin, Chuyang; Irie, Yasuhiko; Storek, Kelly M.; Yang, Jaeun Jane; Siehnel, Richard ...; Howell, P. Lynne; Singh, Pradeep K ...; Tolker-Nielsen, ...; Parsek, Matthew ...; Schweizer, Herbe ...; Harrison, Joe J.; show less...

About the subject

MEDICAL AND HEALTH SCIENCES: MEDICAL AND HEAL ...; and Basic Medicine; and Microbiology in ...

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