Abstract
While Tn-Seq is a powerful tool to determine genome-wide bacterial fitness in high-throughput, culturing transposon-mutant libraries in pools can mask community or other complex single-cell phenotypes. Droplet Tn-Seq (dTn-Seq) solves this problem by microfluidics facilitated encapsulation of individual transposon mutants into growth medium-in-oil droplets, thereby enabling isolated growth, free from the influence of the population. Here we describe and validate microfluidic chip design, production, encapsulation, and dTn-Seq sample preparation. We determine that 1–3% of mutants in Streptococcus pneumoniae have a different fitness when grown in isolation and show how dTn-Seq can help identify leads for gene function, including those involved in hyper-competence, processing of alpha-1-acid glycoprotein, sensitivity against the human leukocyte elastase and microcolony formation. Additionally, we show dTn-Seq compatibility with microscopy, FACS and investigations of bacterial cell-to-cell and bacteria-host cell interactions. dTn-Seq reduces costs and retains the advantages of Tn-Seq, while expanding the method’s original applicability.
Highlights
While Transposon-insertion sequencing (Tn-Seq) is a powerful tool to determine genome-wide bacterial fitness in highthroughput, culturing transposon-mutant libraries in pools can mask community or other complex single-cell phenotypes
Besides the ability to resolve complex single-cell behavior, droplets have additional advantages and applications including a substantial reduction in culture media volume, it enables analysis of bacterial microcolony formation, interbacterial interactions, and bacterial–host cell interactions through fluorescence-activated cell sorting (FACS) and microscopy
To determine the functionality of droplet Tn-Seq (dTn-Seq), six transposon-insertion libraries (~10,000 mutants each) of S. pneumoniae were grown in batch culture as pooled populations (“standard” Tn-Seq) and encapsulated in droplets as single cells under four different conditions: (1) growth medium with glucose as the major carbon source; (2) growth medium with the complex host glycan alpha-1-acid glycoprotein (AGP) as the major carbon source; (3) growth medium with glucose and the human leukocyte produced protease elastase, which is responsible for neutrophil-mediated killing of S. pneumoniae; and (4) growth medium with glucose and a 1% agarose-droplet density to assess how a solid substrate that provides structural support affects single-cell growth and promotes microcolony formation
Summary
While Tn-Seq is a powerful tool to determine genome-wide bacterial fitness in highthroughput, culturing transposon-mutant libraries in pools can mask community or other complex single-cell phenotypes. To assess the fitness of each mutant in isolation from the rest of the pool and thereby possibly uncover population masking effects, a microfluidic device was designed and manufactured in-house that enables encapsulation of individual cells into growth medium-in-oil droplets, in which bacteria can be cultured (Fig. 2a; Supplementary Fig. 1, Supplementary Data 1).
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