Design, modeling and synthesis of an in vitro transcription rate regulatory circuit

This paper describes the design, modeling and realization of a synthetic in vitro circuit that aims at regulating the rate of mRNA transcription. Two DNA templates are designed to interact through their transcripts, creating negative feedback loops that will equate their transcription rates at steady state. A mathematical model is developed for this circuit, consisting of a set of ODEs derived from the mass action laws and Michaelis-Menten kinetics involving all the present chemical species. The DNA strands were accordingly designed, following thermodynamics principles and minimizing unwanted interactions. Preliminary experimental results show that the circuit is performing the expected task, by matching at steady state the transcription rates of the two DNA templates

Design and performance of in vitro transcription rate regulatory circuits

This paper proposes a synthetic in vitro circuit that aims at regulating the rate of RNA transcription through positive feedback interactions. This design is dual to a previously synthesized transcriptional rate regulator based on self-repression. Two DNA templates are designed to interact through their transcripts, creating cross activating feedback loops that will equate their transcription rates at steady state. A mathematical model is developed for this circuit, consisting of a set of ODEs derived from the mass action laws and Michaelis-Menten kinetics involving all the present chemical species. This circuit is then compared to its regulatory counterpart based on negative feedback. A global sensitivity analysis reveals the fundamental features of the two designs by evaluating their equilibrium response to changes in the most crucial parameters of the system

Biophysical evidence for intrinsic disorder in the C-terminal tails of the epidermal growth factor receptor (EGFR) and HER3 receptor tyrosine kinases

The epidermal growth factor receptor (EGFR)/ErbB family of receptor tyrosine kinases includes oncogenes important in the progression of breast and other cancers, and they are targets for many drug development strategies. Each member of the ErbB family possesses a unique, structurally uncharacterized C-terminal tail that plays an important role in autophosphorylation and signal propagation. To determine whether these C-terminal tails are intrinsically disordered regions, we conducted a battery of biophysical experiments on the EGFR and HER3 tails. Using hydrogen/deuterium exchange mass spectrometry, we measured the conformational dynamics of intracellular half constructs and compared the tails with the ordered kinase domains. The C-terminal tails demonstrate more rapid deuterium exchange behavior when compared with the kinase domains. Next, we expressed and purified EGFR and HER3 tail-only constructs. Results from circular dichroism spectroscopy, size exclusion chromatography with multiangle light scattering, dynamic light scattering, analytical ultracentrifugation, and small angle X-ray scattering each provide evidence that the EGFR and HER3 C-terminal tails are intrinsically disordered with extended, non-globular structure in solution. The intrinsic disorder and extended conformation of these tails may be important for their function by increasing the capture radius and reducing the thermodynamic barriers for binding of downstream signaling proteins

Tuning a synthetic in vitro oscillator using control-theoretic tools

This paper demonstrates the effectiveness of simple control-theoretic tools in generating simulation-guided experiments on a synthetic in vitro oscillator. A theoretical analysis of the behavior of such system is motivated by high cost, time consuming experiments, together with the excessive number of tuning parameters. A simplified model of the synthetic oscillator is chosen to capture only its essential features. The model is analyzed using the small gain theorem and the theory of describing functions. Such analysis reveals what are the parameters that primarily determine when the system can admit stable oscillations. Experimental verification of the theoretical and numerical findings is carried out and confirms the predicted results regarding the role of production and degradation rates

A Multi-Model Approach to Identification of Biosynthetic Pathways

We present an identification framework for biochemical systems that allows multiple candidate models to be compared. This framework is designed to select a model that fits the data while maintaining model simplicity. The model identification task is divided into a parameter estimation stage and a model comparison stage. Model selection is based on calculating Akaike's information criterion, which is a systematic method for determining the model that best represents a set of experimental data. Two case studies are presented: a simulated transcriptional control circuit and a system of oscillators that has been built and characterized in vitro. In both examples the multi-model framework is able to discriminate between model candidates to select the one that best describes the data

Design of insulating devices for in vitro synthetic circuits

This paper describes a synthetic in vitro genetic circuit programmed to work as an insulating device. This circuit is composed of nucleic acids, which can be designed to interact according to user defined rules, and of few proteins that perform catalytic functions. A model of the circuit is derived from first principle biochemical laws. This model is shown to exhibit time-scale separation that makes its output insensitive to downstream time varying loads. Simulation results show the circuit effectiveness and represent the starting point for future experimental testing of the device

An analytical approach to bistable biological circuit discrimination using real algebraic geometry

Biomolecular circuits with two distinct and stable steady states have been identified as essential components in a wide range of biological networks, with a variety of mechanisms and topologies giving rise to their important bistable property. Understanding the differences between circuit implementations is an important question, particularly for the synthetic biologist faced with determining which bistable circuit design out of many is best for their specific application. In this work we explore the applicability of Sturm's theorem—a tool from nineteenth-century real algebraic geometry—to comparing ‘functionally equivalent’ bistable circuits without the need for numerical simulation. We first consider two genetic toggle variants and two different positive feedback circuits, and show how specific topological properties present in each type of circuit can serve to increase the size of the regions of parameter space in which they function as switches. We then demonstrate that a single competitive monomeric activator added to a purely monomeric (and otherwise monostable) mutual repressor circuit is sufficient for bistability. Finally, we compare our approach with the Routh–Hurwitz method and derive consistent, yet more powerful, parametric conditions. The predictive power and ease of use of Sturm's theorem demonstrated in this work suggest that algebraic geometric techniques may be underused in biomolecular circuit analysis

Bistable State Switch Enables Ultrasensitive Feedback Control in Heterogeneous Microbial Populations

Molecular feedback control circuits can improve robustness of gene expression at the single cell level. This achievement can be offset by requirements of rapid protein expression, that may induce cellular stress, known as burden, that reduces colony growth. To begin to address this challenge we take inspiration by 'division-of-labor' in heterogeneous cell populations: we propose to combine bistable switches and quorum sensing systems to coordinate gene expression at the population level. We show that bistable switches in individual cells operating in parallel yield an ultrasensitive response, while cells maintain heterogeneous levels of gene expression to avoid burden across all cells. Within a feedback loop, these switches can achieve robust reference tracking and adaptation to disturbances at the population-level. We also demonstrate that molecular sequestration enables tunable hysteresis in individual switches, making it possible to obtain a wide range of stable population-level expressions

Design and performance of in vitro transcription rate regulatory circuits

This paper proposes a synthetic in vitro circuit that aims at regulating the rate of RNA transcription through positive feedback interactions. This design is dual to a previously synthesized transcriptional rate regulator based on self-repression. Two DNA templates are designed to interact through their transcripts, creating cross activating feedback loops that will equate their transcription rates at steady state. A mathematical model is developed for this circuit, consisting of a set of ODEs derived from the mass action laws and Michaelis-Menten kinetics involving all the present chemical species. This circuit is then compared to its regulatory counterpart based on negative feedback. A global sensitivity analysis reveals the fundamental features of the two designs by evaluating their equilibrium response to changes in the most crucial parameters of the system

Tuning a synthetic in vitro oscillator using control-theoretic tools

This paper demonstrates the effectiveness of simple control-theoretic tools in generating simulation-guided experiments on a synthetic in vitro oscillator. A theoretical analysis of the behavior of such system is motivated by high cost, time consuming experiments, together with the excessive number of tuning parameters. A simplified model of the synthetic oscillator is chosen to capture only its essential features. The model is analyzed using the small gain theorem and the theory of describing functions. Such analysis reveals what are the parameters that primarily determine when the system can admit stable oscillations. Experimental verification of the theoretical and numerical findings is carried out and confirms the predicted results regarding the role of production and degradation rates