Randomly barcoded transposon mutant libraries for gut commensals I: Strategies for efficient library construction
Surya Tripathi,
Carlos Geert Pieter Voogdt,
Stefan Oliver Bassler,
Mary Anderson,
Po-Hsun Huang,
Nazgul Sakenova,
Tümay Capraz,
Sunit Jain,
Alexandra Koumoutsi,
Afonso Martins Bravo,
Valentine Trotter,
Michael Zimmerman,
Justin L. Sonnenburg,
Cullen Buie,
Athanasios Typas,
Adam M. Deutschbauer,
Anthony L. Shiver,
Kerwyn Casey Huang
Affiliations
Surya Tripathi
Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA 94720, USA
Carlos Geert Pieter Voogdt
Genome Biology Unit, EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany; Structural and Computational Biology Unit, EMBL Meyerhofstraße 1, 69117 Heidelberg, Germany
Stefan Oliver Bassler
Genome Biology Unit, EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany; Faculty of Biosciences, Heidelberg University, Grabengasse 1, 69117 Heidelberg, Germany
Mary Anderson
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Po-Hsun Huang
Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Nazgul Sakenova
Genome Biology Unit, EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany
Tümay Capraz
Genome Biology Unit, EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany; Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Sunit Jain
Chan Zuckerberg Biohub, San Francisco, CA 94158, USA
Alexandra Koumoutsi
Genome Biology Unit, EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany
Afonso Martins Bravo
Department of Fundamental Microbiology, University of Lausanne, 1015 Lausanne, Switzerland
Valentine Trotter
Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
Michael Zimmerman
Structural and Computational Biology Unit, EMBL Meyerhofstraße 1, 69117 Heidelberg, Germany
Justin L. Sonnenburg
Chan Zuckerberg Biohub, San Francisco, CA 94158, USA; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
Cullen Buie
Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Athanasios Typas
Genome Biology Unit, EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany; Structural and Computational Biology Unit, EMBL Meyerhofstraße 1, 69117 Heidelberg, Germany; Corresponding author
Adam M. Deutschbauer
Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Corresponding author
Anthony L. Shiver
Department of Bioengineering, Stanford University, Stanford, CA 94305, USA; Corresponding author
Kerwyn Casey Huang
Chan Zuckerberg Biohub, San Francisco, CA 94158, USA; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Bioengineering, Stanford University, Stanford, CA 94305, USA; Corresponding author
Summary: Randomly barcoded transposon mutant libraries are powerful tools for studying gene function and organization, assessing gene essentiality and pathways, discovering potential therapeutic targets, and understanding the physiology of gut bacteria and their interactions with the host. However, construction of high-quality libraries with uniform representation can be challenging. In this review, we survey various strategies for barcoded library construction, including transposition systems, methods of transposon delivery, optimal library size, and transconjugant selection schemes. We discuss the advantages and limitations of each approach, as well as factors to consider when selecting a strategy. In addition, we highlight experimental and computational advances in arraying condensed libraries from mutant pools. We focus on examples of successful library construction in gut bacteria and their application to gene function studies and drug discovery. Given the need for understanding gene function and organization in gut bacteria, we provide a comprehensive guide for researchers to construct randomly barcoded transposon mutant libraries.
