SorTn-seq

SorTn-seq maps regulatory determinants of gene expression in bacteria by combining saturation Tn5-like transposon mutagenesis, promoter-driven fluorescent reporters, fluorescence-activated cell sorting (FACS), deep sequencing of transposon insertion sites, and comparative bioinformatic analysis.

Key Features:

• High-Density Transposon Mutagenesis: Uses a Tn5-like transposon system to introduce high-density, genome-wide insertions.
• Fluorescence-Activated Cell Sorting (FACS): Sorts bacterial cells expressing a promoter-driven fluorescent reporter into fluorescence-intensity bins.
• Deep Sequencing of Insertion Sites: Uses deep sequencing to identify and map transposon insertion sites from each fluorescence bin.
• Bioinformatic Analysis: Compares insertion frequencies across fluorescence bins with existing bioinformatic pipelines and software to identify putative regulators based on enrichment or depletion.
• Library Preparation: DNA preparation for sequencing employs commercial kits supplemented with custom primers to recover transposon–genome junctions.

Scientific Applications:

• Identification of regulators: Identifies genetic elements and regulatory loci that influence expression of a specific promoter-driven reporter.
• Regulatory network mapping: Explores regulatory interactions within bacterial genomes by associating loci with reporter expression changes.
• Genome-wide mutation effects on expression: Investigates how genome-wide transposon insertions affect gene expression levels.

Methodology:

Deep sequencing reads are used to identify and map transposon insertion sites, and existing bioinformatic pipelines and software compare insertion frequencies across fluorescence-sorted bins to identify genomic loci enriched or depleted in specific bins.