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ARS Home » Southeast Area » Stoneville, Mississippi » Genomics and Bioinformatics Research » Research » Research Project #433541

Research Project: Development of Tn-Seq Methods for the Functional Characterization of Bacteria

Location: Genomics and Bioinformatics Research

Project Number: 6066-21310-005-28-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Sep 20, 2017
End Date: Sep 2, 2022

Objective:
Our understanding of the physiology and metabolism of bacteria is based upon our knowledge of gene function and even well-studied bacteria possess 30-50% of genes with unknown or poorly characterized function. Similarly, the number and identity of essential genes in most model bacteria are unknown. To investigate gene function and essentiality, methods such as Transposon Sequencing (TnSeq) have been developed over the past few years. These methods utilize transposon mutant libraries in which the host organism’s genome has been randomly disrupted by a transposon-antibiotic resistance marker. Using next-generation high-throughput sequencing, the locations of these transposons can be precisely mapped. Genes that are underrepresented or absent from the library can be considered essential. Using different growth conditions and medium formulations yields a subset of genes that are essential only under given conditions, and others that are universally essential. Thus, by comparing the essentiality of genes under different conditions we can begin to elucidate the function of poorly understood genes. Applying this methodology to bacteria that are potential plant pathogens could enable high-throughput identification of genes required virulence, by comparing strains to their non-pathogenic relatives. In addition, by applying this methodology to enteric bacteria we may be able to develop better containment strategies. This project will develop methods for construction of TnSeq libraries and build a bioinformatics pipeline to map, and determine the fitness of mutants using sequencing data.

Approach:
1. The cooperator will develop methods to create transposon mutants and generate mutant libraries. 2. The cooperator will screen mutant pools against selective conditions and generate samples for mapping and quantification. 3. ARS and the cooperator will jointly Sequence TnSeq samples. 4. ARS and the cooperator will jointly develop bioinformatics workflows for the automated analysis of TnSeq libraries and process TnSeq samples.