Abstract
Transposon sequencing, or Tn-seq, combines transposon mutagenesis and massively parallel sequencing to allow for rapid and high-throughput identification of genes that play roles in fitness within environments of interest. The bacterial pathogen Vibrio cholerae is an excellent candidate for Tn-seq screens due to the availability of a plasmid-based in vivo transposition system and the relative ease with which the cholera disease state can be modeled in animals. This chapter will describe a method for performing Tn-seq screens on V. cholerae in the infant rabbit model of cholera.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
van Opijnen T, Bodi KL, Camilli A (2009) Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6:767–772. https://doi.org/10.1038/nmeth.1377
van Opijnen T, Camilli A (2013) Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat Rev Microbiol 11:435–442. https://doi.org/10.1038/nrmicro3033
van Opijnen T, Lazinski DW, Camilli A (2014) Genome-wide fitness and genetic interactions determined by Tn-seq, a high-throughput massively parallel sequencing method for microorganisms. Curr Protoc Mol Biol 106:7.16.1–7.1624. https://doi.org/10.1002/0471142727.mb0716s106
Lazinski DW, Camilli A (2013) Homopolymer tail-mediated ligation PCR: a streamlined and highly efficient method for DNA cloning and library construction. BioTechniques 54:25–34. https://doi.org/10.2144/000113981
McDonough E, Lazinski DW, Camilli A (2014) Identification of in vivo regulators of the Vibrio cholerae xds gene using a high-throughput genetic selection. Mol Microbiol 92:302–315. https://doi.org/10.1111/mmi.12557
Klein BA, Tenorio EL, Lazinski DW et al (2012) Identification of essential genes of the periodontal pathogen Porphyromonas gingivalis. BMC Genomics 13:578. https://doi.org/10.1186/1471-2164-13-578
Bender J, Kleckner N (1992) Tn10 insertion specificity is strongly dependent upon sequences immediately adjacent to the target-site consensus sequence. Proc Natl Acad Sci U S A 89:7996–8000
Kamp HD, Patimalla-Dipali B, Lazinski DW et al (2013) Gene fitness landscapes of Vibrio cholerae at important stages of its life cycle. PLoS Pathog 9:e1003800. https://doi.org/10.1371/journal.ppat.1003800
Ujiiye A, Nakatomi M, Utsunomiya A et al (1968) Experimental cholera in mice: I. First report on the oral infection. Trop Med 10:65–71
Klose KE (2000) The suckling mouse model of cholera. Trends Microbiol 8:189–191. https://doi.org/10.1016/S0966-842X(00)01721-2
Ritchie JM, Rui H, Bronson RT, Waldor MK (2010) Back to the future: studying cholera pathogenesis using infant rabbits. mBio 1:e00047-10. https://doi.org/10.1128/mBio.00047-10
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Shull, L.M., Camilli, A. (2018). Transposon Sequencing of Vibrio cholerae in the Infant Rabbit Model of Cholera. In: Sikora, A. (eds) Vibrio Cholerae. Methods in Molecular Biology, vol 1839. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8685-9_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8685-9_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8684-2
Online ISBN: 978-1-4939-8685-9
eBook Packages: Springer Protocols