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Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing
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Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing

DOI:
10.3791/61612-v

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08:19 min

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July 07, 2020

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, ,
1Department of Biology,University of Texas at Arlington

Chapters

  • 00:04Introduction
  • 00:57Bacterial Mating
  • 02:47Generation of Final Bacterial Mutant Library
  • 04:11DNA Shearing and Poly-C Tail Addition
  • 06:23Results: Transposon Mutant Library
  • 07:31Conclusion

Summary

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We describe a method to generate saturating transposon mutant libraries in Gram-negative bacteria and subsequent preparation of DNA amplicon libraries for high-throughput sequencing. As an example, we focus on the ESKAPE pathogen, Acinetobacter baumannii, but this protocol is amenable to a wide range of Gram-negative organisms.

Tags

Keywords: Transposon Insertion LibraryGram-negative BacteriaHigh-throughput SequencingEssential GenesAntibiotic ResistanceIn Vivo FitnessMechanical ShearingPoly-CTLRestriction EnzymesAdapter LigationBacterial GeneticsGenotype-phenotype RelationshipsMatingDiaminopimelic AcidGlycerolFrozen Stock

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