Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 109, Issue 42, Pages 16817-16822Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1205693109
Keywords
gene network optimization; evolvability; synthetic biology
Categories
Funding
- National Science Foundation (NSF) [0832773]
- NSF
- Direct For Computer & Info Scie & Enginr
- Division of Computing and Communication Foundations [832824] Funding Source: National Science Foundation
- Division of Computing and Communication Foundations
- Direct For Computer & Info Scie & Enginr [0832773] Funding Source: National Science Foundation
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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.
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