Generation of Barcode-Tagged Vibrio fischeri Deletion Strains and Barcode Sequencing (BarSeq) for Multiplex Strain Competitions

Vibrio fischeri is a model mutualist for studying molecular processes affecting microbial colonization of animal hosts. We present a detailed protocol for a barcode sequencing (BarSeq) approach that combines targeted gene deletion with short-read sequencing technology to enable studies of mixed bacterial populations. This protocol includes wet lab steps to plan and produce the deletions, approaches to scale up mutant generation, protocols to prepare and conduct the strain competition, library preparation for sequencing on an Illumina iSeq 100 instrument, and data analysis with the barseq python package. Aspects of this protocol could be readily adapted for tagging wild-type V. fischeri strains with a neutral barcode for examination of population dynamics or BarSeq analyses in other species. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Production of the erm-bar DNA

Basic Protocol 2: Generation of a targeted and barcoded deletion strain of V. fischeri

Alternate Protocol: Parallel generation of multiple barcode-tagged V. fischeri deletion strains

Basic Protocol 3: Setting up mixed populations of barcode-tagged strains

Basic Protocol 4: Performing a competitive growth assay

Basic Protocol 5: Amplicon library preparation and equimolar pooling

Basic Protocol 6: Sequencing on Illumina iSeq 100

Basic Protocol 7: BarSeq data analysis