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Rapid ordering of barcoded transposon insertion libraries of anaerobic bacteria

Nature Protocols volume 16, pages 3049–3071 (2021)Cite this article

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Abstract

Commensal bacteria from the human intestinal microbiota play important roles in health and disease. Research into the mechanisms by which these bacteria exert their effects is hampered by the complexity of the microbiota, the strict growth requirements of the individual species and a lack of genetic tools and resources. The assembly of ordered transposon insertion libraries, in which nearly all nonessential genes have been disrupted and the strains stored as independent monocultures, would be a transformative resource for research into many microbiota members. However, assembly of these libraries must be fast and inexpensive in order to empower investigation of the large number of species that typically compose gut communities. The methods used to generate ordered libraries must also be adapted to the anaerobic growth requirements of most intestinal bacteria. We have developed a protocol to assemble ordered libraries of transposon insertion mutants that is fast, cheap and effective for even strict anaerobes. The protocol differs from currently available methods by making use of cell sorting to order the library and barcoded transposons to facilitate the localization of ordered mutations in the library. By tracking transposon insertions using barcode sequencing, our approach increases the accuracy and reduces the time and effort required to locate mutants in the library. Ordered libraries can be sorted and characterized over the course of 2 weeks using this approach. We expect this protocol will lower the barrier to generating comprehensive, ordered mutant libraries for many species in the human microbiota, allowing for new investigations into genotype–phenotype relationships within this important microbial ecosystem.

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Fig. 1: Schematic of the entire process of sorting the transposon-insertion library and mapping the barcodes.
Fig. 2: Analysis workflow to locate mutant strains in the ordered library.
Fig. 3: Sorting schematic.
Fig. 4: Cryostock creation schematic.
Fig. 5: Schematic of pooling strategy.
Fig. 6: Statistics of the 40-plate ordered library.

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Data availability

The sequencing data used in this study are publicly available on NCBI as part of BioProject PRJNA573294. The sequence reads from the Bar-seq experiment on pools of the ordered library are available as part of BioSample SAMN12807646. The sequence reads from the RB-TnSeq experiment on the ordered library are available as part of BioSample SAMN12809978. Plate reader output files, microscopy images and extracted growth parameters that support the findings of this study (Supplementary Figs. 2–4) are available from the corresponding author upon request.

Code availability

The code required for locating insertion mutants in ordered libraries is available on BitBucket (https://bitbucket.org/kchuanglab/resolve_barcode_position/src). These scripts rely on previously published code available on BitBucket (https://bitbucket.org/berkeleylab/feba/src). The code in this protocol has been peer reviewed.

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Acknowledgements

The authors thank the Huang lab for helpful discussions. This work was supported in part by the Allen Discovery Center at Stanford University on Systems Modeling of Infection (to K.C.H.) and NIH grants RM1 GM135102 (to A.M.D. and K.C.H.) and R01 AI147023 (to K.C.H.). K.C.H. is a Chan Zuckerberg Biohub Investigator. The authors also acknowledge the hospitality of the Aspen Center for Physics, which is supported by NSF grant PHY-1607611. The following reagents were obtained through BEI Resources, NIAID, NIH as part of the Human Microbiome Project: Lactobacillus reuteri, strain CF48-3A, HM-102; Clostridium innocuum, strain 6_1_30, HM-173; Clostridium symbiosum, strain WAL-14163, HM-309; Bacteroides finegoldii, strain CL09T03C10, HM-727; Parabacteroides johnsonii, strain CL02T12C29, HM-731.

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Authors and Affiliations

  1. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Anthony L. Shiver & Kerwyn Casey Huang

  2. Department of Genetics, Stanford University, Stanford, CA, USA

    Rebecca Culver

  3. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Adam M. Deutschbauer

  4. Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA, USA

    Adam M. Deutschbauer

  5. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA

    Kerwyn Casey Huang

  6. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Kerwyn Casey Huang

Authors
  1. Anthony L. Shiver
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  2. Rebecca Culver
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  3. Adam M. Deutschbauer
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  4. Kerwyn Casey Huang
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Contributions

A.L.S., A.M.D. and K.C.H. conceived the study. A.L.S. designed experiments and collected data. A.L.S. and R.C. analyzed the data. A.L.S., R.C., A.M.D. and K.C.H. interpreted the data and wrote the manuscript.

Corresponding author

Correspondence to Kerwyn Casey Huang.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Buz Barstow and Matthew Waldor for their contribution to the peer review of this work.

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Key references using this protocol

Price, M. N. et al. Nature 557, 503 (2018): https://doi.org/10.1038/s41586-018-0124-0

Liu, H. et al. Cell Rep. 34, 108789 (2021): https://doi.org/10.1016/j.celrep.2021.108789

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Supplementary Information

Supplementary Figs. 1–5, Supplementary Results and Supplementary Tables 1–3.

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Shiver, A.L., Culver, R., Deutschbauer, A.M. et al. Rapid ordering of barcoded transposon insertion libraries of anaerobic bacteria. Nat Protoc 16, 3049–3071 (2021). https://doi.org/10.1038/s41596-021-00531-3

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