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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    • "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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    • "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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