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    Construction of a large-scale Burkholderia cenocepacia J2315 transposon mutant library

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    Type
    Conference Paper
    Authors
    Wong, Yee-Chin
    Pain, Arnab cc
    Nathan, Sheila
    KAUST Department
    Biological and Environmental Sciences and Engineering (BESE) Division
    Bioscience Program
    Date
    2014-09-04
    Online Publication Date
    2014-09-04
    Print Publication Date
    2014
    Permanent link to this record
    http://hdl.handle.net/10754/652853
    
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    Abstract
    Burkholderia cenocepacia, a pathogenic member of the Burkholderia cepacia complex (Bcc), has emerged as a significant threat towards cystic fibrosis patients, where infection often leads to the fatal clinical manifestation known as cepacia syndrome. Many studies have investigated the pathogenicity of B. cenocepacia as well as its ability to become highly resistant towards many of the antibiotics currently in use. In addition, studies have also been undertaken to understand the pathogen's capacity to adapt and survive in a broad range of environments. Transposon based mutagenesis has been widely used in creating insertional knock-out mutants and coupled with recent advances in sequencing technology, robust tools to study gene function in a genome-wide manner have been developed based on the assembly of saturated transposon mutant libraries. In this study, we describe the construction of a large-scale library of B. cenocepacia transposon mutants. To create transposon mutants of B. cenocepacia strain J2315, electrocompetent bacteria were electrotransformed with the EZ-Tn5 <TET-1> transposome. Tetracyline resistant colonies were harvested off selective agar and pooled. Mutants were generated in multiple batches with each batch consisting of ~ 20,000 to 40,000 mutants. Transposon insertion was validated by PCR amplification of the transposon region. In conclusion, a saturated B. cenocepacia J2315 transposon mutant library with an estimated total number of 500,000 mutants was successfully constructed. This mutant library can now be further exploited as a genetic tool to assess the function of every gene in the genome, facilitating the discovery of genes important for bacterial survival and adaptation, as well as virulence.
    Citation
    Wong Y-C, Pain A, Nathan S (2014) Construction of a large-scale Burkholderia cenocepacia J2315 transposon mutant library. Available: http://dx.doi.org/10.1063/1.4895267.
    Sponsors
    This project was funded by the Ministry of Science, Technology and Innovation, Malaysia (MOSTI). Yee-Chin Wong was supported by the MyBrain15 scholarship from the Ministry of Higher Education Malaysia.
    Publisher
    AIP Publishing
    Conference/Event name
    2014 Postgraduate Colloquium of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology, UKM FST 2014
    DOI
    10.1063/1.4895267
    Additional Links
    https://aip.scitation.org/doi/abs/10.1063/1.4895267
    ae974a485f413a2113503eed53cd6c53
    10.1063/1.4895267
    Scopus Count
    Collections
    Conference Papers; Biological and Environmental Science and Engineering (BESE) Division; Bioscience Program

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