Efficient electrotransformation of Bacteroides fragilis

Minoru Ichimura, Haruyuki Nakayama-Imaohji, Shin Wakimoto, Hidetoshi Morita, Tetsuya Hayashi, Tomomi Kuwahara

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

This study describes refined electroporation parameters for efficient transformation of Bacteroides fragilis by plasmids prepared from laboratory strains of Escherichia coli. Development of the method used included determination of the optimal growth conditions for competent cell preparation, selectable antimicrobial resistance markers, electric field strength, and postpulse incubation time. Of the four E. coli-Bacteroides shuttle plasmids tested (pVAL-1, pVAL-2, pNLYl, and pLYL05), pLYL05 containing the cefoxitin resistance marker was found to be the most suitable for B. fragilis transformation, and it generated 2- to 900-fold more transformants (about 104 transformants per μg pLYL05 DNA) than the other plasmids. For the 72-h cultivation period tested, B. fragilis cells harvested at 48 h yielded the highest numbers of transformants. The transformation efficiency of pLYL05 increased linearly with the electric field strength over a range from 5.0 to 12.5 kV/cm. At least 3 h of postpulse incubation was required to maximize the transformation efficiency. For deletion of B. fragilis genes by homologous recombination, competent cells grown to early exponential phase and 12 h of postpuise incubation were required for efficient integration of the pLYL05-based suicide vector into the target site. The expected integration was obtained in B. fragilis strain NCTC9343 only when a homologously prepared (i.e., in vivo methylated) suicide vector was used. Spontaneous resolution of the diploid successfully deleted the expected genetic region. Our simple and efficient plasmid transfer method enabled disruption of a B. fragilis gene using in vivo-methylated targeted vectors. Our optimized electroporation parameters provide a useful tool for genetic manipulation of Bacteroides species.

Original languageEnglish
Pages (from-to)3325-3332
Number of pages8
JournalApplied and Environmental Microbiology
Volume76
Issue number10
DOIs
Publication statusPublished - May 2010
Externally publishedYes

Fingerprint

Bacteroides fragilis
plasmid
suicide
incubation
plasmids
Plasmids
electric field
Bacteroides
Electroporation
electroporation
Suicide
gene
Escherichia coli
recombination
Cefoxitin
Homologous Recombination
homologous recombination
cells
fold
Diploidy

ASJC Scopus subject areas

  • Food Science
  • Biotechnology
  • Ecology
  • Applied Microbiology and Biotechnology

Cite this

Ichimura, M., Nakayama-Imaohji, H., Wakimoto, S., Morita, H., Hayashi, T., & Kuwahara, T. (2010). Efficient electrotransformation of Bacteroides fragilis. Applied and Environmental Microbiology, 76(10), 3325-3332. https://doi.org/10.1128/AEM.02420-09

Efficient electrotransformation of Bacteroides fragilis. / Ichimura, Minoru; Nakayama-Imaohji, Haruyuki; Wakimoto, Shin; Morita, Hidetoshi; Hayashi, Tetsuya; Kuwahara, Tomomi.

In: Applied and Environmental Microbiology, Vol. 76, No. 10, 05.2010, p. 3325-3332.

Research output: Contribution to journalArticle

Ichimura, M, Nakayama-Imaohji, H, Wakimoto, S, Morita, H, Hayashi, T & Kuwahara, T 2010, 'Efficient electrotransformation of Bacteroides fragilis', Applied and Environmental Microbiology, vol. 76, no. 10, pp. 3325-3332. https://doi.org/10.1128/AEM.02420-09
Ichimura M, Nakayama-Imaohji H, Wakimoto S, Morita H, Hayashi T, Kuwahara T. Efficient electrotransformation of Bacteroides fragilis. Applied and Environmental Microbiology. 2010 May;76(10):3325-3332. https://doi.org/10.1128/AEM.02420-09
Ichimura, Minoru ; Nakayama-Imaohji, Haruyuki ; Wakimoto, Shin ; Morita, Hidetoshi ; Hayashi, Tetsuya ; Kuwahara, Tomomi. / Efficient electrotransformation of Bacteroides fragilis. In: Applied and Environmental Microbiology. 2010 ; Vol. 76, No. 10. pp. 3325-3332.
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