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Bacterial genetic engineering by means of recombineering for reverse genetics

Ursula Fels (UGent) , Kris Gevaert (UGent) and Petra Van Damme (UGent)
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  • PROPHECY (PROPHECY: Translational control in infection biology: riboproteogenomics of bacterial pathogens)
Abstract
Serving a robust platform for reverse genetics enabling the in vivo study of gene functions primarily in enterobacteriaceae, recombineering -or recombination-mediated genetic engineering- represents a powerful and relative straightforward genetic engineering tool. Catalyzed by components of bacteriophage-encoded homologous recombination systems and only requiring short ~40 to 50 base homologies, the targeted and precise introduction of modifications (e.g., deletions, knockouts, insertions and point mutations) into the chromosome and other episomal replicons is empowered. Furthermore, by its ability to make use of both double- and single-stranded linear DNA editing substrates (e.g., PCR products or oligonucleotides, respectively), lengthy subcloning of specific DNA sequences is circumvented. Further, the more recent implementation of CRISPR-associated endonucleases has allowed for more efficient screening of successful recombinants by the selective purging of non-edited cells, as well as the creation of markerless and scarless mutants. In this review we discuss various recombineering strategies to promote different types of gene modifications, how they are best applied, and their possible pitfalls.
Keywords
bacterial genetics, phage-based homologous recombination, precise genome editing, recombineering, selection markers, Reverse Genetics, enterobactaeriaceae, ESCHERICHIA-COLI GENOME, ONE-STEP INACTIVATION, RECOMBINATION SYSTEM, HOMOLOGOUS RECOMBINATION, MEDIATED RECOMBINATION, BACTERIOPHAGE-LAMBDA, POSITIVE SELECTION, RED RECOMBINATION, MULTIPLE GENES, HOST STRAINS

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Citation

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MLA
Fels, Ursula, et al. “Bacterial Genetic Engineering by Means of Recombineering for Reverse Genetics.” FRONTIERS IN MICROBIOLOGY, vol. 11, 2020, doi:10.3389/fmicb.2020.548410.
APA
Fels, U., Gevaert, K., & Van Damme, P. (2020). Bacterial genetic engineering by means of recombineering for reverse genetics. FRONTIERS IN MICROBIOLOGY, 11. https://doi.org/10.3389/fmicb.2020.548410
Chicago author-date
Fels, Ursula, Kris Gevaert, and Petra Van Damme. 2020. “Bacterial Genetic Engineering by Means of Recombineering for Reverse Genetics.” FRONTIERS IN MICROBIOLOGY 11. https://doi.org/10.3389/fmicb.2020.548410.
Chicago author-date (all authors)
Fels, Ursula, Kris Gevaert, and Petra Van Damme. 2020. “Bacterial Genetic Engineering by Means of Recombineering for Reverse Genetics.” FRONTIERS IN MICROBIOLOGY 11. doi:10.3389/fmicb.2020.548410.
Vancouver
1.
Fels U, Gevaert K, Van Damme P. Bacterial genetic engineering by means of recombineering for reverse genetics. FRONTIERS IN MICROBIOLOGY. 2020;11.
IEEE
[1]
U. Fels, K. Gevaert, and P. Van Damme, “Bacterial genetic engineering by means of recombineering for reverse genetics,” FRONTIERS IN MICROBIOLOGY, vol. 11, 2020.
@article{8671808,
  abstract     = {Serving a robust platform for reverse genetics enabling the in vivo study of gene functions primarily in enterobacteriaceae, recombineering -or recombination-mediated genetic engineering- represents a powerful and relative straightforward genetic engineering tool. Catalyzed by components of bacteriophage-encoded homologous recombination systems and only requiring short ~40 to 50 base homologies, the targeted and precise introduction of modifications (e.g., deletions, knockouts, insertions and point mutations) into the chromosome and other episomal replicons is empowered. Furthermore, by its ability to make use of both double- and single-stranded linear DNA editing substrates (e.g., PCR products or oligonucleotides, respectively), lengthy subcloning of specific DNA sequences is circumvented. Further, the more recent implementation of CRISPR-associated endonucleases has allowed for more efficient screening of successful recombinants by the selective purging of non-edited cells, as well as the creation of markerless and scarless mutants. In this review we discuss various recombineering strategies to promote different types of gene modifications, how they are best applied, and their possible pitfalls.},
  articleno    = {548410},
  author       = {Fels, Ursula and Gevaert, Kris and Van Damme, Petra},
  issn         = {1664-302X},
  journal      = {FRONTIERS IN MICROBIOLOGY},
  keywords     = {bacterial genetics,phage-based homologous recombination,precise genome editing,recombineering,selection markers,Reverse Genetics,enterobactaeriaceae,ESCHERICHIA-COLI GENOME,ONE-STEP INACTIVATION,RECOMBINATION SYSTEM,HOMOLOGOUS RECOMBINATION,MEDIATED RECOMBINATION,BACTERIOPHAGE-LAMBDA,POSITIVE SELECTION,RED RECOMBINATION,MULTIPLE GENES,HOST STRAINS},
  language     = {eng},
  title        = {Bacterial genetic engineering by means of recombineering for reverse genetics},
  url          = {http://dx.doi.org/10.3389/fmicb.2020.548410},
  volume       = {11},
  year         = {2020},
}

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