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

Yee Chin Wong, Arnab Pain, Sheila Nathan

Research output: Chapter in Book/Report/Conference proceedingConference contribution

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.

Original languageEnglish
Title of host publication2014 UKM FST Postgraduate Colloquium - Proceedings of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology 2014 Postgraduate Colloquium
EditorsZahari Ibrahim, Haja Maideen Kader Maideen, Nazlina Ibrahim, Nurul Huda Abd Karim, Taufik Yusof, Fatimah Abdul Razak, Nurulkamal Maseran, Rozida Mohd Khalid, Noor Baa'yah Ibrahim, Hasidah Mohd. Sidek, Mohd Salmi Md Noorani, Norbert Simon
PublisherAmerican Institute of Physics Inc.
Pages583-586
Number of pages4
ISBN (Electronic)9780735412507
DOIs
Publication statusPublished - 1 Jan 2014
Event2014 Postgraduate Colloquium of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology, UKM FST 2014 - Selangor, Malaysia
Duration: 9 Apr 201411 Apr 2014

Publication series

NameAIP Conference Proceedings
Volume1614
ISSN (Print)0094-243X
ISSN (Electronic)1551-7616

Other

Other2014 Postgraduate Colloquium of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology, UKM FST 2014
CountryMalaysia
CitySelangor
Period9/4/1411/4/14

Fingerprint

genes
genome
cystic fibrosis
virulence
mutagenesis
sequencing
pathogens
antibiotics
infectious diseases
bacteria
insertion
assembly

Keywords

  • Burkholderia cenocepacia
  • Burkholderia cepacia complex
  • Mutant
  • Transposon

ASJC Scopus subject areas

  • Physics and Astronomy(all)

Cite this

Wong, Y. C., Pain, A., & Nathan, S. (2014). Construction of a large-scale burkholderia cenocepacia J2315 transposon mutant library. In Z. Ibrahim, H. M. K. Maideen, N. Ibrahim, N. H. A. Karim, T. Yusof, F. A. Razak, N. Maseran, R. M. Khalid, N. B. Ibrahim, H. M. Sidek, M. S. M. Noorani, ... N. Simon (Eds.), 2014 UKM FST Postgraduate Colloquium - Proceedings of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology 2014 Postgraduate Colloquium (pp. 583-586). (AIP Conference Proceedings; Vol. 1614). American Institute of Physics Inc.. https://doi.org/10.1063/1.4895267

Construction of a large-scale burkholderia cenocepacia J2315 transposon mutant library. / Wong, Yee Chin; Pain, Arnab; Nathan, Sheila.

2014 UKM FST Postgraduate Colloquium - Proceedings of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology 2014 Postgraduate Colloquium. ed. / Zahari Ibrahim; Haja Maideen Kader Maideen; Nazlina Ibrahim; Nurul Huda Abd Karim; Taufik Yusof; Fatimah Abdul Razak; Nurulkamal Maseran; Rozida Mohd Khalid; Noor Baa'yah Ibrahim; Hasidah Mohd. Sidek; Mohd Salmi Md Noorani; Norbert Simon. American Institute of Physics Inc., 2014. p. 583-586 (AIP Conference Proceedings; Vol. 1614).

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Wong, YC, Pain, A & Nathan, S 2014, Construction of a large-scale burkholderia cenocepacia J2315 transposon mutant library. in Z Ibrahim, HMK Maideen, N Ibrahim, NHA Karim, T Yusof, FA Razak, N Maseran, RM Khalid, NB Ibrahim, HM Sidek, MSM Noorani & N Simon (eds), 2014 UKM FST Postgraduate Colloquium - Proceedings of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology 2014 Postgraduate Colloquium. AIP Conference Proceedings, vol. 1614, American Institute of Physics Inc., pp. 583-586, 2014 Postgraduate Colloquium of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology, UKM FST 2014, Selangor, Malaysia, 9/4/14. https://doi.org/10.1063/1.4895267
Wong YC, Pain A, Nathan S. Construction of a large-scale burkholderia cenocepacia J2315 transposon mutant library. In Ibrahim Z, Maideen HMK, Ibrahim N, Karim NHA, Yusof T, Razak FA, Maseran N, Khalid RM, Ibrahim NB, Sidek HM, Noorani MSM, Simon N, editors, 2014 UKM FST Postgraduate Colloquium - Proceedings of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology 2014 Postgraduate Colloquium. American Institute of Physics Inc. 2014. p. 583-586. (AIP Conference Proceedings). https://doi.org/10.1063/1.4895267
Wong, Yee Chin ; Pain, Arnab ; Nathan, Sheila. / Construction of a large-scale burkholderia cenocepacia J2315 transposon mutant library. 2014 UKM FST Postgraduate Colloquium - Proceedings of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology 2014 Postgraduate Colloquium. editor / Zahari Ibrahim ; Haja Maideen Kader Maideen ; Nazlina Ibrahim ; Nurul Huda Abd Karim ; Taufik Yusof ; Fatimah Abdul Razak ; Nurulkamal Maseran ; Rozida Mohd Khalid ; Noor Baa'yah Ibrahim ; Hasidah Mohd. Sidek ; Mohd Salmi Md Noorani ; Norbert Simon. American Institute of Physics Inc., 2014. pp. 583-586 (AIP Conference Proceedings).
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