droplet-Tn-Seq combines microfluidics with Tn-Seq identifying complex single-cell phenotypes

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 solves that problem by microfluidics facilitated encapsulation of individual transposon mutants into liquid-in-oil droplets, thereby enabling isolated growth, free from the influence of the population. Importantly, all advantages of Tn-Seq are conserved, while reducing costs and greatly extending its applicability.

Functional genetics of human gut commensal Bacteroides thetaiotaomicron reveals metabolic requirements for growth across environments

“…Second, the PUL component(s) may perform its activity extracellularly, such that other mutants in the pooled library can complement any growth deficiency in trans. In addition to assaying mutants individually, approaches for performing genome-wide fitness assays within droplets hold the potential for eliminating complementation by other mutants (Thibault et al, 2019). Third, some PULs may not be expressed under our growth conditions or are involved in the breakdown of polysaccharides that we did not profile.…”

Bacterial ClpP Protease Is a Potential Target for Methyl Gallate

“…The transposon insertion of RS_RS01640 resulted in an increase in relative fitness under the treatment. However, the RS_RS01640 -deleted mutant did not show obvious resistance to MG. One possibility is that the growth attenuation caused by the deletion of RS_RS01640 masked the resistance to MG, and a more sensitive method is needed to confirm the effect of RS_RS01640 on MG. Another possibility is that all transposon-inserted mutants were cultured en masse in a pool, and the relative abundance of a certain mutant might be affected by other mutants or by public goods, which makes the results from Tn-seq differ from the phenotypes of each individual ( Thibault et al, 2019 ).…”

The Genetic Basis of phage susceptibility, cross-resistance and host-range in Salmonella

“…Considering differences in phage infectivity in a panel of strains with highly conserved genetic determinants, we posit that systematic study of differences in transcriptional and translation processes in these strains might provide more insights as illustrated in a few recent phage-host interaction studies(Brandão et al, 2021; Howard-Varona et al, 2018). Future studies could also employ recently developed methods(Mutalik et al, 2019; Rishi et al, 2020; Thibault et al, 2019) to provide higher resolution into phage-host interactions and may aid in filling the knowledge gaps on phage host-range. These methods could be extended to a few closely related and phylogenetically distant strains to understand the variability in host factors impacting phage infectivity patterns.…”