Abstract
RNA regulators are emerging as powerful tools to engineer synthetic genetic networks or rewire existing ones. A potential strength of RNA networks is that they may be able to propagate signals on time scales that are set by the fast degradation rates of RNAs. However, a current bottleneck to verifying this potential is the slow design-build-test cycle of evaluating these networks in vivo. Here, we adapt an Escherichia coli-based cell-free transcription-translation (TX-TL) system for rapidly prototyping RNA networks. We used this system to measure the response time of an RNA transcription cascade to be approximately five minutes per step of the cascade. We also show that this response time can be adjusted with temperature and regulator threshold tuning. Finally, we use TX-TL to prototype a new RNA network, an RNA single input module, and show that this network temporally stages the expression of two genes in vivo.
Highlights
A central goal of synthetic biology is to control cellular behavior in a predictable manner.[1]
In order to assess the feasibility of using TX-TL for characterizing RNA circuitry, we first tested the basic functionality of the central regulator in our RNA cascade, the pT181 transcriptional attenuator[19] (Figure 2, Att-1)
We found that an attenuator plasmid concentration of 0.5 nM struck a balance between fluorescence signal and DNA concentration, and this concentration was used in subsequent experiments
Summary
A central goal of synthetic biology is to control cellular behavior in a predictable manner.[1]. Genetic networks webs of interactions between cellular regulatory molecules are responsible for dynamically turning these genes on at the right time and place and, in effect, are the circuitry that implement behavioral programs in cells.[2] Because of this, a central focus of synthetic biology has been to control cellular behavior by engineering genetic networks from the bottom up.[1]. Fluorescent proteins are generally used as reporters, monitoring fluorescence over time allows the characterization of circuit dynamics Because of their simplicity, cell-free reactions reduce the time for testing a constructed genetic circuit design from days to as little as an hour.[33,36] Since these systems do not require selection markers or DNA replication to maintain circuitry constructs, there are no limitations on DNA circularization or on plasmid origin of replication and antibiotic compatibility.[33,36] This flexibility allows for faster, multiplexed generation of circuit constructs, further reducing design-buildtest cycle times. Since cell-free reactions lack a membrane, DNA encoding different regulators can be added at any time during the reactions, enabling the rapid characterization of network response as a function of perturbations that are extremely difficult or even impossible to do inside cells[34]
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