title: Fine-tuning gene networks using simple sequence repeats.
abstract: The parameters in a complex synthetic gene network must be extensively tuned before the network functions as designed. Here, we introduce a simple and general approach to rapidly tune gene networks in Escherichia coli using hypermutable simple sequence repeats embedded in the spacer region of the ribosome binding site. By varying repeat length, we generated expression libraries that incrementally and predictably sample gene expression levels over a 1,000-fold range. We demonstrate the utility of the approach by creating a bistable switch library that programmatically samples the expression space to balance the two states of the switch, and we illustrate the need for tuning by showing that the switch's behavior is sensitive to host context. Further, we show that mutation rates of the repeats are controllable in vivo for stability or for targeted mutagenesis--suggesting a new approach to optimizing gene networks via directed evolution. This tuning methodology should accelerate the process of engineering functionally complex gene networks.
authors.affiliation: Department of Electrical Engineering, University of Washington, Seattle, WA 98195, USA.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Egbert
authors.forename: Eric
authors.initials: E
authors.lastname: Klavins
mesh_terms: D021903:DNA, Ribosomal Spacer / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D053263:Gene Regulatory Networks / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D018895:Microsatellite Repeats / Q000235:genetics*; D059645:Mutation Rate
chemical_list: D021903:DNA, Ribosomal Spacer