Fine-tuning gene networks using simple sequence repeats.

Department of Electrical Engineering, University of Washington, Seattle, WA 98195.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 08/2012; 109(42):16817-22. DOI: 10.1073/pnas.1205693109
Source: PubMed

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.

Download full-text


Available from: Eric Klavins, Jul 04, 2014
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Inducible expression is a versatile genetic tool for controlling gene transcription, determining gene functions and other uses. Herein, we describe our attempts to create several inducible systems based on a cumate or a resorcinol switch, a hammerhead ribozyme, the LacI repressor, and isopropyl β-d-thiogalactopyranoside (IPTG). We successfully developed a new cumate (p-isopropylbenzoic acid)-inducible gene switch in actinobacteria that is based on the CymR regulator, the operator sequence (cmt) from the Pseudomonas putida cumate degradation operon and P21 synthetic promoter. Resorcinol-inducible expression system is also functional and is composed of the RolR regulator and the PA3 promoter fused with the operator (rolO) from the Corynebacterium glutamicum resorcinol catabolic operon. Using the gusA (β-glucuronidase) gene as a reporter, we showed that the newly generated expression systems are tightly regulated and hyper-inducible. The activity of the uninduced promoters is negligible in both cases. Whereas the induction factor reaches 45 for Streptomyces albus in the case of cumate switch and 33 in the case of resorcinol toggle. The systems are also dose-dependent, which allows the modulation of gene expression even from a single promoter. In addition, the cumate system is versatile, given that it is functional in different actinomycetes. Finally, these systems are nontoxic and inexpensive, as these are characteristics of cumate and resorcinol, and they are easy to use because inducers are water-soluble and easily penetrate cells. Therefore, the P21-cmt-CymR and PA3-rolO-RolR systems are powerful tools for engineering actinobacteria.
    Applied Microbiology and Biotechnology 07/2014; 98(20). DOI:10.1007/s00253-014-5918-x · 3.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Synthetic Biology is the "Engineering of Biology" - it aims to use a forward-engineering design cycle based on specifications, modelling, analysis, experimental implementation, testing and validation to modify natural or design new, synthetic biology systems so that they behave in a predictable fashion. Motivated by the need for truly plug-and-play synthetic biological components we present a comprehensive review of ways in which the various parts of a biological system can be modified systematically. In particular, we review the list of 'dials' that are available to the designer and discuss how they can be modelled, tuned and implemented. The dials are categorized according to whether they operate at the global, transcriptional, translational or post translational level and the resolution that they operate at. We end this review with a discussion on the relative advantages and disadvantages of some dials over others.
    Microbiology 05/2013; 159(Pt 7). DOI:10.1099/mic.0.067975-0 · 2.84 Impact Factor
  • Source
    Proceedings of the National Academy of Sciences 10/2012; 109(42):16758-9. DOI:10.1073/pnas.1214118109 · 9.81 Impact Factor