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: 9.67). 08/2012; 109(42):16817-22. DOI: 10.1073/pnas.1205693109
Source: PubMed


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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Available from: Eric Klavins, Jul 04, 2014
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    • "Synthetic biology tends to apply known genetic engineering approaches to construct new organisms with desired properties . Currently, a plethora of synthetic genetic modules are being developed, such as promoters, ribosomal binding sites (RBSs), terminators, transfer RNAs (tRNAs), riboswitches, and ribozymes (Salis et al. 2009; Lucks et al. 2011; Egbert and Klavins 2012; Keasling 2012; Siegl et al. 2013; Rudolph et al. 2013). Combining these synthetic " BioBricks " allows the directed evolution of biological systems with the goal of adapting existing components for novel functions. "
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    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 · 3.34 Impact Factor
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    • "Similar to DNA, polyA RNA sequences are less flexible than mixed or polyU RNA sequences (50,51). Differences in RNA rigidity have been shown to alter the ribosome’s binding affinity to spacer regions separating the SD and start codon sequences (52); binding free energy differences are ∼2.5 kcal/mol between polyA and polyAU spacer sequences, and ∼5 kcal/mol between polyA and polyU spacer sequences. Incorporating a sequence-dependent model for RNA rigidity into the distortion penalty calculations could potentially increase their accuracy. "
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    ABSTRACT: The ribosome's interactions with mRNA govern its translation rate and the effects of post-transcriptional regulation. Long, structured 5' untranslated regions (5' UTRs) are commonly found in bacterial mRNAs, though the physical mechanisms that determine how the ribosome binds these upstream regions remain poorly defined. Here, we systematically investigate the ribosome's interactions with structured standby sites, upstream of Shine-Dalgarno sequences, and show that these interactions can modulate translation initiation rates by over 100-fold. We find that an mRNA's translation initiation rate is controlled by the amount of single-stranded surface area, the partial unfolding of RNA structures to minimize the ribosome's binding free energy penalty, the absence of cooperative binding and the potential for ribosomal sliding. We develop a biophysical model employing thermodynamic first principles and a four-parameter free energy model to accurately predict the ribosome's translation initiation rates for 136 synthetic 5' UTRs with large structures, diverse shapes and multiple standby site modules. The model predicts and experiments confirm that the ribosome can readily bind distant standby site modules that support high translation rates, providing a physical mechanism for observed context effects and long-range post-transcriptional regulation.
    Nucleic Acids Research 11/2013; 42(4). DOI:10.1093/nar/gkt1139 · 9.11 Impact Factor
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    • "Regulated processes such as promoter repression or activation have also shown growth rate dependence, affecting genetic networks as a result (Klumpp et al., 2009; Scott et al., 2010; Tan et al., 2009). There are a wide range of strains and organisms that can be used to harbour synthetic genetic networks, and in some cases, the networks work predictably across different strains (Prindle et al., 2012) whilst in other cases the behaviour of the network can be drastically altered by changing the host cell (Egbert & Klavins, 2012). In this latter reference, two strains of E. coli were used that both contained a LacID mutation but with slightly different genotypes, exemplifying that even similar strains can have profound effects on network function. "
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    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 · 2.56 Impact Factor
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