Abstract
Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach for discovering the functions of bacterial genes. However, the development of a suitable TnSeq strategy for a given bacterium can be costly and time-consuming. To meet this challenge, we describe a part-based strategy for constructing libraries of hundreds of transposon delivery vectors, which we term "magic pools." Within a magic pool, each transposon vector has a different combination of upstream sequences (promoters and ribosome binding sites) and antibiotic resistance markers as well as a random DNA barcode sequence, which allows the tracking of each vector during mutagenesis experiments. To identify an efficient vector for a given bacterium, we mutagenize it with a magic pool and sequence the resulting insertions; we then use this efficient vector to generate a large mutant library. We used the magic pool strategy to construct transposon mutant libraries in five genera of bacteria, including three genera of the phylum Bacteroidetes. IMPORTANCE Molecular genetics is indispensable for interrogating the physiology of bacteria. However, the development of a functional genetic system for any given bacterium can be time-consuming. Here, we present a streamlined approach for identifying an effective transposon mutagenesis system for a new bacterium. Our strategy first involves the construction of hundreds of different transposon vector variants, which we term a "magic pool." The efficacy of each vector in a magic pool is monitored in parallel using a unique DNA barcode that is introduced into each vector design. Using archived DNA "parts," we next reassemble an effective vector for making a whole-genome transposon mutant library that is suitable for large-scale interrogation of gene function using competitive growth assays. Here, we demonstrate the utility of the magic pool system to make mutant libraries in five genera of bacteria.
Highlights
Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach for discovering the functions of bacterial genes
A number of TnSeq-like variants have been described; these variants include transposon-directed insertion sequencing (TraDIS) [9], high-throughput insertion tracking by deep sequencing (HITS) [10], insertion sequencing (IN-Seq) [11], and rapid transposon liquid enrichment sequencing (TnLE-seq) [12]
In this study, we present a strategy for testing the efficacy of hundreds of transposon vectors in parallel against a target bacterium
Summary
Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach for discovering the functions of bacterial genes. The development of a suitable TnSeq strategy for a given bacterium can be costly and time-consuming To meet this challenge, we describe a part-based strategy for constructing libraries of hundreds of transposon delivery vectors, which we term “magic pools.”. Accelerating genetic tool development in bacteria using a combinatorial approach msystems.asm.org 1 pooled together, and the relative abundance of all of the mutant strains can be monitored by next-generation sequencing of the genomic DNA flanking the transposon insertion. By constructing very large libraries of mutants containing thousands to millions of unique insertion strains, the genome-wide interrogation of mutant fitness and gene fitness can be conducted in parallel in a single tube The first such strategy, termed TnSeq (transposon mutagenesis coupled to next-generation sequencing), was used in Streptococcus pneumoniae to globally measure single-gene fitness and to screen genetic interactions [8]. A number of TnSeq-like variants have been described; these variants include transposon-directed insertion sequencing (TraDIS) [9], high-throughput insertion tracking by deep sequencing (HITS) [10], insertion sequencing (IN-Seq) [11], and rapid transposon liquid enrichment sequencing (TnLE-seq) [12]
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