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Development of a counterselectable transposon to create markerless knockouts from an 18,432-clone ordered Mycobacterium bovis bacillus Calmette-Guérin mutant resource

Katlyn Borgers (UGent) , Kristof Vandewalle (UGent) , Annelies Van Hecke (UGent) , Gitte Michielsen (UGent) , Evelyn Plets (UGent) , Loes van Schie (UGent) , Sandrine Vanmarcke (UGent) , Laurent Schindfessel (UGent) , Nele Festjens (UGent) and Nico Callewaert (UGent)
(2020) MSYSTEMS. 5(4).
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Abstract
Mutant resources are essential to improve our understanding of the biology of slow-growing mycobacteria, which include the causative agents of tuberculosis in various species, including humans. The generation of deletion mutants in slow-growing mycobacteria in a gene-by-gene approach in order to make genome-wide ordered mutant resources is still a laborious and costly approach, despite the recent development of improved methods. On the other hand, transposon mutagenesis in combination with Cartesian pooling-coordinate sequencing (CP-CSeq) allows the creation of large archived Mycobacterium transposon insertion libraries. However, such mutants contain selection marker genes with a risk of polar gene effects, which are undesired both for research and for use of these mutants as live attenuated vaccines. In this paper, a derivative of the Himar1 transposon is described which allows the generation of clean, markerless knockouts from archived transposon libraries. By incorporating FRT sites for FlpE/FRT-mediated recombination and I-SceI sites for ISceIM-based transposon removal, we enable two thoroughly experimentally validated possibilities to create unmarked mutants from such marked transposon mutants. The FRT approach is highly efficient but leaves an FRT scar in the genome, whereas the I-SceI-mediated approach can create mutants without any heterologous DNA in the genome. The combined use of CP-CSeq and this optimized transposon was applied in the BCG Danish 1331 vaccine strain (WHO reference 07/270), creating the largest ordered, characterized resource of mutants in a member of the Mycobacterium tuberculosis complex (18,432 clones, mutating 83% of the nonessential M. tuberculosis homologues), from which markerless knockouts can be easily generated.
Keywords
Mycobacterium tuberculosis complex, characterized transposon library, optimized Himar1 transposon, transposon mutagenesis, unmarked mutants, I-SCEI MEGANUCLEASE, EFFICIENT TRANSGENESIS, GENE DELETIONS, TUBERCULOSIS, RECOMBINASE, EXPRESSION, MUTAGENESIS, REPAIR

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MLA
Borgers, Katlyn, et al. “Development of a Counterselectable Transposon to Create Markerless Knockouts from an 18,432-Clone Ordered Mycobacterium Bovis Bacillus Calmette-Guérin Mutant Resource.” MSYSTEMS, vol. 5, no. 4, 2020, doi:10.1128/msystems.00180-20.
APA
Borgers, K., Vandewalle, K., Van Hecke, A., Michielsen, G., Plets, E., van Schie, L., … Callewaert, N. (2020). Development of a counterselectable transposon to create markerless knockouts from an 18,432-clone ordered Mycobacterium bovis bacillus Calmette-Guérin mutant resource. MSYSTEMS, 5(4). https://doi.org/10.1128/msystems.00180-20
Chicago author-date
Borgers, Katlyn, Kristof Vandewalle, Annelies Van Hecke, Gitte Michielsen, Evelyn Plets, Loes van Schie, Sandrine Vanmarcke, Laurent Schindfessel, Nele Festjens, and Nico Callewaert. 2020. “Development of a Counterselectable Transposon to Create Markerless Knockouts from an 18,432-Clone Ordered Mycobacterium Bovis Bacillus Calmette-Guérin Mutant Resource.” MSYSTEMS 5 (4). https://doi.org/10.1128/msystems.00180-20.
Chicago author-date (all authors)
Borgers, Katlyn, Kristof Vandewalle, Annelies Van Hecke, Gitte Michielsen, Evelyn Plets, Loes van Schie, Sandrine Vanmarcke, Laurent Schindfessel, Nele Festjens, and Nico Callewaert. 2020. “Development of a Counterselectable Transposon to Create Markerless Knockouts from an 18,432-Clone Ordered Mycobacterium Bovis Bacillus Calmette-Guérin Mutant Resource.” MSYSTEMS 5 (4). doi:10.1128/msystems.00180-20.
Vancouver
1.
Borgers K, Vandewalle K, Van Hecke A, Michielsen G, Plets E, van Schie L, et al. Development of a counterselectable transposon to create markerless knockouts from an 18,432-clone ordered Mycobacterium bovis bacillus Calmette-Guérin mutant resource. MSYSTEMS. 2020;5(4).
IEEE
[1]
K. Borgers et al., “Development of a counterselectable transposon to create markerless knockouts from an 18,432-clone ordered Mycobacterium bovis bacillus Calmette-Guérin mutant resource,” MSYSTEMS, vol. 5, no. 4, 2020.
@article{8672664,
  abstract     = {Mutant resources are essential to improve our understanding of the biology of slow-growing mycobacteria, which include the causative agents of tuberculosis in various species, including humans. The generation of deletion mutants in slow-growing mycobacteria in a gene-by-gene approach in order to make genome-wide ordered mutant resources is still a laborious and costly approach, despite the recent development of improved methods. On the other hand, transposon mutagenesis in combination with Cartesian pooling-coordinate sequencing (CP-CSeq) allows the creation of large archived Mycobacterium transposon insertion libraries. However, such mutants contain selection marker genes with a risk of polar gene effects, which are undesired both for research and for use of these mutants as live attenuated vaccines. In this paper, a derivative of the Himar1 transposon is described which allows the generation of clean, markerless knockouts from archived transposon libraries. By incorporating FRT sites for FlpE/FRT-mediated recombination and I-SceI sites for ISceIM-based transposon removal, we enable two thoroughly experimentally validated possibilities to create unmarked mutants from such marked transposon mutants. The FRT approach is highly efficient but leaves an FRT scar in the genome, whereas the I-SceI-mediated approach can create mutants without any heterologous DNA in the genome. The combined use of CP-CSeq and this optimized transposon was applied in the BCG Danish 1331 vaccine strain (WHO reference 07/270), creating the largest ordered, characterized resource of mutants in a member of the Mycobacterium tuberculosis complex (18,432 clones, mutating 83% of the nonessential M. tuberculosis homologues), from which markerless knockouts can be easily generated.},
  articleno    = {e00180-20},
  author       = {Borgers, Katlyn and Vandewalle, Kristof and Van Hecke, Annelies and Michielsen, Gitte and Plets, Evelyn and van Schie, Loes and Vanmarcke, Sandrine and Schindfessel, Laurent and Festjens, Nele and Callewaert, Nico},
  issn         = {2379-5077},
  journal      = {MSYSTEMS},
  keywords     = {Mycobacterium tuberculosis complex,characterized transposon library,optimized Himar1 transposon,transposon mutagenesis,unmarked mutants,I-SCEI MEGANUCLEASE,EFFICIENT TRANSGENESIS,GENE DELETIONS,TUBERCULOSIS,RECOMBINASE,EXPRESSION,MUTAGENESIS,REPAIR},
  language     = {eng},
  number       = {4},
  pages        = {15},
  title        = {Development of a counterselectable transposon to create markerless knockouts from an 18,432-clone ordered Mycobacterium bovis bacillus Calmette-Guérin mutant resource},
  url          = {http://dx.doi.org/10.1128/msystems.00180-20},
  volume       = {5},
  year         = {2020},
}

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