Bistability of an In Vitro Synthetic Autoregulatory Switch

📝 Abstract

The construction of synthetic biochemical circuits is an essential step for developing quantitative understanding of information processing in natural organisms. Here, we report construction and analysis of an in vitro circuit with positive autoregulation that consists of just four synthetic DNA strands and three enzymes, bacteriophage T7 RNA polymerase, Escherichia coli ribonuclease (RNase) H, and RNase R. The modularity of the DNA switch template allowed a rational design of a synthetic DNA switch regulated by its RNA output acting as a transcription activator. We verified that the thermodynamic and kinetic constraints dictated by the sequence design criteria were enough to experimentally achieve the intended dynamics: a transcription activator configured to regulate its own production. Although only RNase H is necessary to achieve bistability of switch states, RNase R is necessary to maintain stable RNA signal levels and to control incomplete degradation products. A simple mathematical model was used to fit ensemble parameters for the training set of experimental results and was then directly applied to predict time-courses of switch dynamics and sensitivity to parameter variations with reasonable agreement. The positive autoregulation switches can be used to provide constant input signals and store outputs of biochemical networks and are potentially useful for chemical control applications.

💡 Analysis

The construction of synthetic biochemical circuits is an essential step for developing quantitative understanding of information processing in natural organisms. Here, we report construction and analysis of an in vitro circuit with positive autoregulation that consists of just four synthetic DNA strands and three enzymes, bacteriophage T7 RNA polymerase, Escherichia coli ribonuclease (RNase) H, and RNase R. The modularity of the DNA switch template allowed a rational design of a synthetic DNA switch regulated by its RNA output acting as a transcription activator. We verified that the thermodynamic and kinetic constraints dictated by the sequence design criteria were enough to experimentally achieve the intended dynamics: a transcription activator configured to regulate its own production. Although only RNase H is necessary to achieve bistability of switch states, RNase R is necessary to maintain stable RNA signal levels and to control incomplete degradation products. A simple mathematical model was used to fit ensemble parameters for the training set of experimental results and was then directly applied to predict time-courses of switch dynamics and sensitivity to parameter variations with reasonable agreement. The positive autoregulation switches can be used to provide constant input signals and store outputs of biochemical networks and are potentially useful for chemical control applications.

📄 Content

arXiv:1101.0723v1 [q-bio.MN] 4 Jan 2011 Bistability of an In Vitro Synthetic Autoregulatory Switch Pakpoom Subsoontorn1, Jongmin Kim1,4, Erik Winfree2,3,4* Departments of 1 Biology, 2 Computation and Neural Systems, 3 Computer Science and 4 Bioengineering California Institute of Technology {pakpoom,jongmin,winfree}@dna.caltech.edu September 25, 2018 Abstract: The construction of synthetic biochemical circuits is an essential step for developing quantitative understanding of information processing in natural organisms. Here, we report construction and analysis of an in vitro circuit with positive autoregulation that consists of just four synthetic DNA strands and three enzymes, bacteriophage T7 RNA polymerase, Escherichia coli ribonuclease (RNase) H, and RNase R. The modularity of the DNA switch template allowed a rational design of a synthetic DNA switch regulated by its RNA output acting as a transcription activator. We verified that the thermodynamic and kinetic constraints dictated by the sequence design criteria were enough to experimentally achieve the intended dynamics: a transcription activator configured to regulate its own production. Although only RNase H is necessary to achieve bistability of switch states, RNase R is necessary to maintain stable RNA signal levels and to control incomplete degradation products. A simple mathematical model was used to fit ensemble parameters for the training set of experimental results and was then directly applied to predict time-courses of switch dynamics and sensitivity to parameter variations with reasonable agreement. The positive autoregulation switches can be used to provide constant input signals and store outputs of biochemical networks and are potentially useful for chemical control applications. 1 Introduction Within a living cell lies an information processing system: the genetic circuits, the community of genes that regulate one another, and thus allow the cell to express the right genes at the right times. Synthetic biology provides a new approach to understand design principles underlying intricate and dynamic behaviors of natural genetic circuits, by building and analyzing synthetic circuits which exhibit analogous behaviors; this also lays the foundation for future engineering of complex chemical and biological systems. With such circuits, it is possible to test hypotheses by construc- tion, and often synthetic simplicity facilitates quanti- tative analysis as well as systematic engineering de- sign [8, 1, 5, 12]. For designing and constructing synthetic biochem- ical networks, several decision steps are necessary to choose the regulatory molecules and the biochemical in- frastructure that supports network operation. Protein- based synthetic circuits can take advantage of the huge diversity of protein structures and functions that al- lows a wide range of possible regulatory features [5, 1, 7, 2, 32]. Still, from an engineering perspective, it remains a challenge to rationally design a new regula- tory protein with desirable function. RNA-based reg- ulation is an alternative approach for controlling gene expressions [15, 3, 14, 34]. RNA structures and inter- actions with other nucleic acid species can be reliably predicted based on Watson–Crick base-pairing, much more so than typical protein-protein or protein-DNA interactions of protein regulators. As for the choice of biochemical infrastructure, the unintended interactions between the circuit and its environment can be greatly reduced by reconstructing the circuit in vitro. In vitro implementation of efficient transcription and transla- tion machinery [30, 17] for synthetic networks have been successfully implemented [26]. Yet, a supporting envi- ronment for an in vitro RNA-based regulatory circuit can be even simpler as there is no need for translation, protein maturation, and protein-DNA interactions. Previous work [19, 20] introduced in vitro transcrip- tional circuits as simplified synthetic genetic regulatory circuits. Individual switches functioning as inverters and a bistable feedback circuit composed of two in- verters have been demonstrated (Figure 1A, top). Our “DNA switch”, a simplified gene, has a promoter for T7 RNA polymerase (RNAP) flanked by two separate do- mains, an input domain and an output domain. Down- stream of a promoter lies an output domain that en- codes “an RNA product”; on the opposite side of the promoter, an input domain regulated by “an RNA reg- ulator” via simple Watson-Crick base-pairing rule is lo- cated. The modularity of a DNA switch allows for an independent design of an RNA product and an RNA regulator within a switch. Hence, one can “wire” sev- eral switches together to compose a complex regulatory network, in principle, by simply designing the RNA output of one switch to be the RNA regulator of the other switch. Moreover, individual switch characteris- tics such as switching thresholds and maximum output levels are set by the concentrations of switch compo- nen

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