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Analysis of PHB Metabolism Applying Tn5 Mutagenesis in Ralstonia eutropha

  • Matthias Raberg
  • Daniel Heinrich
  • Alexander SteinbüchelEmail author
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Transposon mutagenesis presents a powerful and practicable method to generate single-gene disruption mutants of microorganisms. As naturally occurring transposons “jump” within the genome, molecular biology uses plasmid-bound transposons, which randomly disrupt genomic regions of the target organism. Obtained transposon mutants help to elucidate metabolic pathways and to identify essential genes, which are involved in syntheses or degradation of compounds or are important for other cell processes or cell structures. The best-known transposon, Tn5, codes for different antibiotic resistances as well as for a transposase mediating transposition and a transposase inhibitor protein. A notable example of applied Tn5 mutagenesis is the identification and localization of genes, which are involved in the synthesis of the industrially relevant biopolymer poly(3-hydroxybutyrate) (PHB) in Ralstonia eutropha H16. PHB is synthesized in a three-step pathway, and the key genes of R. eutropha were found to be organized as a single operon. In this chapter, the generation and analysis of Tn5-induced mutants of R. eutropha is described. This procedure starts with the transfer of the Tn5-harboring plasmid pSUP5011 into R. eutropha by conjugation, is followed by the screening of mutants defective in PHB accumulation, and is then completed by identifying genes, which have been disrupted by Tn5 by sequence analyses.

Keywords:

Conjugation PHB metabolism Ralstonia eutropha Suicide plasmid technique Tn5 mutagenesis Two-step gene walking 

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Matthias Raberg
    • 1
  • Daniel Heinrich
    • 1
  • Alexander Steinbüchel
    • 1
    • 2
    Email author
  1. 1.Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-UniversitätMünsterGermany
  2. 2.Environmental Sciences DepartmentKing Abdulaziz UniversityJeddahSaudi Arabia

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