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

doi:10.1016/j.celrep.2023.113517

Randomly barcoded transposon mutant libraries for gut commensals I: Strategies for efficient library construction

Cell Rep. 2024 Jan 23;43(1):113517. doi: 10.1016/j.celrep.2023.113517. Epub 2023 Dec 23.

Authors

Surya Tripathi¹, Carlos Geert Pieter Voogdt², Stefan Oliver Bassler³, Mary Anderson⁴, Po-Hsun Huang⁵, Nazgul Sakenova⁶, Tümay Capraz⁷, Sunit Jain⁸, Alexandra Koumoutsi⁶, Afonso Martins Bravo⁹, Valentine Trotter¹⁰, Michael Zimmerman¹¹, Justin L Sonnenburg¹², Cullen Buie⁵, Athanasios Typas¹³, Adam M Deutschbauer¹⁴, Anthony L Shiver¹⁵, Kerwyn Casey Huang¹⁶

Affiliations

¹ Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
² Genome Biology Unit, EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany; Structural and Computational Biology Unit, EMBL Meyerhofstraße 1, 69117 Heidelberg, Germany.
³ Genome Biology Unit, EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany; Faculty of Biosciences, Heidelberg University, Grabengasse 1, 69117 Heidelberg, Germany.
⁴ Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁵ Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁶ Genome Biology Unit, EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany.
⁷ Genome Biology Unit, EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany; Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁸ Chan Zuckerberg Biohub, San Francisco, CA 94158, USA.
⁹ Department of Fundamental Microbiology, University of Lausanne, 1015 Lausanne, Switzerland.
¹⁰ Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
¹¹ Structural and Computational Biology Unit, EMBL Meyerhofstraße 1, 69117 Heidelberg, Germany.
¹² Chan Zuckerberg Biohub, San Francisco, CA 94158, USA; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.
¹³ Genome Biology Unit, EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany; Structural and Computational Biology Unit, EMBL Meyerhofstraße 1, 69117 Heidelberg, Germany. Electronic address: typas@embl.de.
¹⁴ 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. Electronic address: amdeutschbauer@lbl.gov.
¹⁵ Department of Bioengineering, Stanford University, Stanford, CA 94305, USA. Electronic address: ashiver@stanford.edu.
¹⁶ 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. Electronic address: kchuang@stanford.edu.

PMID: 38142397
DOI: 10.1016/j.celrep.2023.113517

Abstract

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.

Keywords: CP: Microbiology; RB-Tn-seq; arrayed libraries; functional genomics; gut microbiota; in vitro screening; magic pools; protein function; transposon mutagenesis.

Publication types

Review
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, N.I.H., Extramural
Comment

MeSH terms

Bacteria / genetics
Cloning, Molecular
DNA Transposable Elements* / genetics
Gene Library
High-Throughput Nucleotide Sequencing* / methods
Mutagenesis, Insertional / genetics

Substances

DNA Transposable Elements

Grants and funding

RM1 GM135102/GM/NIGMS NIH HHS/United States