K-132. Phenotypic Characterization of Microorganisms by Barcoded Transposon Mutagenesis

A. Deutschbauer1, J. Oh2, M. Price1, P. Dehal1, D. Bruno2, J. Kuehl1, R. Chakraborty1, T. C. Hazen1, C. Nislow3, G. Giaever3, R. W. Davis2, A. P. Arkin1;
1Lawrence Berkeley Natl. Lab., Berkeley, CA, 2Stanford Genome Technology Ctr., Stanford, CA, 3Univ. of Toronto, Toronto, ON, CANADA.

Systems-level analyses of less studied bacteria are limited by the presence of numerous uncharacterized genes and an over reliance on annotations from well studied bacteria such as E. coli. To meet this challenge, we are developing a mutagenesis and phenotyping strategy that is comprehensive across the genome and applicable to any microorganism amenable to transposon mutagenesis. We have cloned and sequence-verified ~3000 barcode modules into a Gateway entry vector. Each module is a 175 base pair element containing two unique 20 base pair sequences, the UPTAG and DOWNTAG, flanked by common PCR priming sites. Each module can then be rapidly transferred in vitro to any DNA element, such as a transposon, that is made Gateway compatible. Transposon mutants marked by the modules will be sequenced to determine which of the ~3000 barcode modules was used and which gene was disrupted. Transposon mutants can be rapidly re-arrayed into a single pool containing ~3000 uniquely tagged, sequence-verified mutant strains. By sequencing saturating numbers of transposon mutants, we can identify and assay mutants in most nonessential genes in a given genome. The fitness of each mutant in the pool will be monitored in parallel by the hybridization of the barcodes to an Affymetrix microarray containing the barcode complements in a system identical to that used for the yeast deletion collection. Compared to other approaches for the parallel analysis of transposon mutants such as signature tagged mutagenesis, genetic footprinting, and transposon site hybridization, our approach offers higher throughput, a single microarray design is universal for any organism, single mutational events are assayed, and mutant strains are archived for verification, further analysis, and distribution. The completion of this project will enable the quantitative phenotypic analysis of thousands of mutants across a wide range of conditions. In addition, our genetic resources provide a framework for the systematic genetic interrogation of individual pathways. Here we describe the application of this approach to Shewanella oneidensis MR1 and Desulfovibrio desulfuricans G20.