Microarray-based genetic footprinting strategy to identify strain improvement genes after competitive selection of transposon libraries

Methods Mol Biol. 2011;765:83-97. doi: 10.1007/978-1-61779-197-0_6.

Abstract

Successful strain engineering involves perturbing key nodes within the cellular network. How the -network's connectivity affects the phenotype of interest and the ideal nodes to modulate, however, are frequently not readily apparent. To guide the generation of a list of candidate nodes for detailed investigation, designers often examine the behavior of a representative set of strains, such as a library of transposon insertion mutants, in the environment of interest. Here, we first present design principles for creating a maximally informative competitive selection. Then, we describe how to globally quantify the change in distribution of strains within a transposon library in response to a competitive selection by amplifying the DNA adjacent to the transposons and hybridizing it to a microarray. Finally, we detail strategies for analyzing the resulting hybridization data to identify genes and pathways that contribute both negatively and positively to fitness in the desired environment.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • DNA Footprinting*
  • DNA Transposable Elements / genetics*
  • Gene Library*
  • Mutagenesis, Insertional / genetics*
  • Oligonucleotide Array Sequence Analysis*
  • Selection, Genetic*

Substances

  • DNA Transposable Elements