High-throughput, quantitative analyses of genetic interactions in E. coli.
Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor-driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed genetic interaction analysis technology for E. coli (GIANT-coli), permits us to systematically generate and array double-mutant cells on solid media in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli.
Typas
Athanasios
A
Department of Microbiology and Immunology, University of California at San Francisco, 600 16th Street, San Francisco, California 94158, USA.
Nichols
Robert J
RJ
Siegele
Deborah A
DA
Shales
Michael
M
Collins
Sean R
SR
Lim
Bentley
B
Braberg
Hannes
H
Yamamoto
Natsuko
N
Takeuchi
Rikiya
R
Wanner
Barry L
BL
Mori
Hirotada
H
Weissman
Jonathan S
JS
Krogan
Nevan J
NJ
Gross
Carol A
CA
D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000235:genetics*; D016680:Genome, Bacterial; D009154:Mutation