Refined method for the genomic integration of complex synthetic circuits

Abstract

Genetic reconstruction of regulatory gene circuits is currently applied in systematic dynamics and structure-function studies of intact cellular networks in systems biology. We present a modified procedure for the integration of complex genetic circuits into the Escherichia coli genome, to provide an efficient synthetic approach for stochastic study and the artificial engineering of genetic networks. Linear artificial sequences of various lengths were easily integrated into the bacterial genome at one time. Comparison of the cellular concentrations of proteins encoded by genes carried on plasmids or the genome indicated that genome recombination could minimize the copy number noise in the genetic circuit, allowing precise design and interpretation of the cellular network. The refined recombination procedure allowed efficient construction of a single copy of a complex genetic circuit in cells, and the resultant reduced fluctuation in copy number led to accurate phenotypic behaviour of the genome-integrated synthetic switch corresponding to the design principle. The availability of long-fragment insertions makes the reconstruction of complex networks easy on the genome, and provides a powerful tool for precise engineering in synthetic and systems biology.