Negative auto-regulation increases the input dynamic-range of the arabinose system of Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Gene regulation networks are made of recurring regulatory patterns, called network motifs. One of the most common network motifs is negative auto-regulation, in which a transcription factor represses its own production. Negative auto-regulation has several potential functions: it can shorten the response time (time to reach halfway to steady-state), stabilize expression against noise, and linearize the gene's input-output response curve. This latter function of negative auto-regulation, which increases the range of input signals over which downstream genes respond, has been studied by theory and synthetic gene circuits. Here we ask whether negative auto-regulation preserves this function also in the context of a natural system, where it is embedded within many additional interactions. To address this, we studied the negative auto-regulation motif in the arabinose utilization system of <it>Escherichia coli</it>, in which negative auto-regulation is part of a complex regulatory network.</p> <p>Results</p> <p>We find that when negative auto-regulation is disrupted by placing the regulator <it>araC </it>under constitutive expression, the input dynamic range of the arabinose system is reduced by 10-fold. The apparent Hill coefficient of the induction curve changes from about <it>n </it>= 1 with negative auto-regulation, to about <it>n </it>= 2 when it is disrupted. We present a mathematical model that describes how negative auto-regulation can increase input dynamic-range, by coupling the transcription factor protein level to the input signal.</p> <p>Conclusions</p> <p>Here we demonstrate that the negative auto-regulation motif in the native arabinose system of <it>Escherichia coli </it>increases the range of arabinose signals over which the system can respond. In this way, negative auto-regulation may help to increase the input dynamic-range while maintaining the specificity of cooperative regulatory systems. This function may contribute to explaining the common occurrence of negative auto-regulation in biological systems.</p

Designing Metabolite Biosensors and Engineering Genetic Circuits to Regulate Metabolic Pathways

Microbial production of chemicals has provided an attractive alternative to chemical synthesis. A key to make this technology economically viable is to improve titers, productivities, and strain robustness. However, pathway productivities and yields are often limited by metabolic imbalances that inhibit cell growth and chemical production. In contrast, natural metabolic pathways are dynamically regulated according to cellular metabolic status. Dynamic regulation allows cells to adjust metabolite concentrations to optimal levels and avoid wasting carbon and energy. Inspired by nature, synthetic regulatory circuits have shown great promise in improving titers and productivities, because they can balance the metabolism by dynamically adjusting enzyme expression levels according to cellular metabolic status. To engineer synthetic regulatory circuits to improve production, we must design and tune metabolite biosensors, and also understand the metabolic dynamics to identify the optimal regulatory architecture. The research presented here addresses both these key aspects and demonstrates an application of genetic circuits to improve pathway production. Specifically, we develop theories that predict and experimentally validate a coupling between dynamic range and response threshold in transcription factor-based biosensors, and provide design guidelines to orthogonally control the biosensor output and its sensitivity. Next, we develop a malonyl-CoA sensor-actuator and demonstrate its application to engineering a negative feedback circuit to improve fatty acid production. Finally, genetic circuits with various architectures are constructed to study metabolic dynamics, which reveal that negative feedback circuits can dramatically accelerate metabolic dynamics. The findings of this dissertation provide rational design principles for transcription factor-based metabolite biosensors and a systematic understanding of metabolic dynamics under various regulation architectures. They provide valuable tools and knowledge to engineer metabolic circuits to regulate various metabolic pathways, increasing titers, productivities, and yields

Development of novel orthogonal genetic circuits, based on extracytoplasmic function (ECF) σ factors

The synthetic biology field aims to apply the engineering 'design-build-test-learn' cycle for the implementation of synthetic genetic circuits modifying the behavior of biological systems. In order to reach this goal, synthetic biology projects use a set of fully characterized biological parts that subsequently are assembled into complex synthetic circuits following a rational, model-driven design. However, even though the bottom-up design approach represents an optimal starting point to assay the behavior of the synthetic circuits under defined conditions, the rational design of such circuits is often restricted by the limited number of available DNA building blocks. These usually consist only of a handful of transcriptional regulators that additionally are often borrowed from natural biological systems. This, in turn, can lead to cross-reactions between the synthetic circuit and the host cell and eventually to loss of the original circuit function. Thus, one of the challenges in synthetic biology is to design synthetic circuits that perform the designated functions with minor cross-reactions (orthogonality). To overcome the restrictions of the widely used transcriptional regulators, this project aims to apply extracytoplasmic function (ECF) σ factors in the design novel orthogonal synthetic circuits. ECFs are the smallest and simplest alternative σ factors that recognize highly specific promoters. ECFs represent one of the most important mechanisms of signal transduction in bacteria, indeed, their activity is often controlled by anti-σ factors. Even though it was shown that the overexpression of heterologous anti-σ factors can generate an adverse effect on cell growth, they represent an attractive solution to control ECF activity. Finally, to date, we know thousands of ECF σ factors, widespread among different bacterial phyla, that are identifiable together with the cognate promoters and anti-σ factors, using bioinformatic approaches. All the above-mentioned features make ECF σ factors optimal candidates as core orthogonal regulators for the design of novel synthetic circuits. In this project, in order to establish ECF σ factors as standard building blocks in the synthetic biology field, we first established a high throughput experimental setup. This relies on microplate reader experiments performed using a highly sensitive luminescent reporter system. Luminescent reporters have a superior signal-to-noise ratio when compared to fluorescent reporters since they do not suffer from the high auto-fluorescence background of the bacterial cell. However, they also have a drawback represented by the constant light emission that can generate undesired cross-talk between neighboring wells on a microplate. To overcome this limitation, we developed a computational algorithm that corrects for luminescence bleed-through and estimates the “true” luminescence activity for each well of a microplate. We show that the correcting algorithm preserves low-level signals close to the background and that it is universally applicable to different experimental conditions. In order to simplify the assembly of large ECF-based synthetic circuits, we designed an ECF toolbox in E. coli. The toolbox allows for the combinatorial assembly of circuits into expression vectors, using a library of reusable genetic parts. Moreover, it also offers the possibility of integrating the newly generated synthetic circuits into four different phage attachment (att) sites present in the genome of E. coli. This allows for a flawless transition between plasmid-encoded and chromosomally integrated genetic circuits, expanding the possible genetic configurations of a given synthetic construct. Moreover, our results demonstrate that the four att sites are orthogonal in terms of the gene expression levels of the synthetic circuits. With the purpose of rationally design ECF-based synthetic circuits and taking advantage of the ECF toolbox, we characterized the dynamic behavior of a set of 15 ECF σ factors, their cognate promoters, and relative anti-σs. Overall, we found that ECFs are non-toxic and functional and that they display different binding affinities for the cognate target promoters. Moreover, our results show that it is possible to optimize the output dynamic range of the ECF-based switches by changing the copy number of the ECFs and target promoters, thus, tuning the input/output signal ratio. Next, by combining up to three ECF-switches, we generated a set of “genetic-timer circuits”, the first synthetic circuits harboring more than one ECF. ECF-based timer circuits sequentially activate a series of target genes with increasing time delays, moreover, the behavior of the circuits can be predicted by a set of mathematical models. In order to improve the dynamic response of the ECF-based constructs, we introduced anti-σ factors in our synthetic circuits. By doing so we first confirmed that anti-σ factors can exert an adverse effect on the growth of E. coli, thus we explored possible solutions. Our results demonstrate that anti-σ factors toxicity can be partially alleviated by generating truncated, soluble variants of the anti-σ factors and, eventually, completely abolished via chromosomal integration of the anti-σ factor-based circuits. Finally, after demonstrating that anti-σ factors can be used to generate a tunable time delay among ECF expression and target promoter activation, we designed ECF/AS-suicide circuits. Such circuits allow for the time-delayed cell-death of E. coli and will serve as a prototype for the further development of ECF/AS-based lysis circuits

Design and Functional Assembly of Synthetic Biological Parts and Devices

Programming living cells with synthetic gene circuits to perform desired tasks has been a major theme in the emerging field of synthetic biology. However, gene circuit engineering currently lacks the same predictability and reliability as seen in other mature engineering disciplines. This thesis focuses on the design and engineering of novel modular and orthogonal biological devices, and the predictable functional assembly of modular biological elements (BioParts) into customisable larger biological devices. The thesis introduces the design methodology for engineering modular and orthogonal biological devices. A set of modular biological devices with digital logic functions, including the AND, NOT and combinatorial NAND gates, were constructed and quantitatively characterised. In particular, a novel genetic AND gate was engineered in Escherichia coli by redesigning the natural HrpR/HrpS heteroregulation motif in the hrp system of Pseudomonas syringae. The AND gate is orthogonal to E. coli chassis, and employs the alternative σ54-dependent gene transcription to achieve tight transcriptional control. Results obtained show that context has a large impact on part and device behaviour, established through the systematic characterisation of a series of biological parts and devices in various biophysical and genetic contexts. A new, effective strategy is presented for the assembly of BioParts into functional customised systems using engineered ‘incontext’ characterised modules aided by modelling, which can significantly increase the predictability of circuit construction by characterising the component parts and modules in the same biophysical and genetic contexts as anticipated in their final systems. Finally, the thesis presents the design and construction of an application-oriented integrated system – the cell density-dependent microbe-based biosensor. The in vivo biosensor was programmed to be able to integrate its own cell density signal through an engineered cell-cell communication module and a second environmental signal through an environment-responsive promoter in the logic AND manner, with GFP as the output readout

Towards engineering microbial consortia using RNA-based genetic controllers

In nature, quorum sensing is a mechanism used by microbes to communicate and coordinate behaviours at the population level. Over the last two decades, synthetic biologists have used the unique property of quorum sensing to sense population density for coordinating cellular behaviours of single and mixed cultures. This gave rise to the development of multicellular biosynthesis systems for metabolic engineering and of spatially distributed systems for synthetic biology. However, robustly controlling the composition of multicellular systems remains a challenge and limits its wide adoption by the metabolic engineering community. Current strategies for controlling synthetic microbial communities vastly rely on engineer- ing metabolic dependencies between microbial species in a process called syntrophy. While syntrophy guarantees the survival of all strains in the coculture, it does not provide a way to control community composition. Existing genetic circuits that can dynamically control community composition often impose too much burden on their hosts for division of labour to be a viable solution to improve yields and titers of valuable metabolic products. Here we investigate the potential of using RNA-based gene circuits to reduce the cost of express- ing heterologous genes for the control of community composition in a two-member E. coli coculture. In this work, we present the development of three genetic modules that rely on RNA species to detect changes in population density and to regulate growth rate when community composition becomes unstable. Together, the modules work in concert to stabilise community composition around a ratio set by the intrinsic properties of the circuit’s genetic components. We identify the key parameters of the circuits that enable tuning of the composition ratio. We characterise the cost of expressing each module of the genetic controller by measuring its impact on the host growth rate and on consumption of free cellular resources. Together these findings highlight the importance of developing host-aware circuits to control community composition so as to enable their wide adoption by metabolic engineers.Open Acces

Genetic Circuits for Transcriptional Regulation in Synechocystis sp. PCC 6803

Microbial biosynthesis has produced a variety of complex compounds using processes that are more environmentally-friendly than many conventional methods. The most common hosts are heterotrophs, which require the addition of an organic carbon source; while cyanobacteria possess many traits that make them a more sustainable biotechnology platform. As phototrophs, cyanobacteria can employ sunlight and carbon dioxide to create many value-added compounds. A wealth of tools has been developed to engineer the commonly used heterotrophs for higher yields and titers; yet, few synthetic biology tools have been designed for cyanobacteria. Furthermore, many of the tools created for heterotrophs do not function as designed in the photosynthetic organisms. We developed a multi-input and several single-input transcriptional regulators for the model cyanobacterium Synechocystis sp. PCC 6803 to address this problem. These circuits were designed to respond to industrially-relevant signals, including oxygen, light and the cells\u27 nitrogen status, in addition to an inexpensive sugar. The two-input AND logic gate we built adds more sophisticated heterologous gene expression to the cyanobacterium\u27s synthetic biology toolbox. The addition of these regulators provides engineers more options when looking for a part that meets the needs of the situation. This was demonstrated by our use of the oxygen-responsive promoter to express, in a heterologous host, genes from a cluster that encodes nitrogenase. This new device can be used to probe the regulation of nitrogen fixation in a photosynthetic cell. Our development of genetic circuits for transcriptional regulation in Synechocystis sp. PCC 6803 improves the viability of this photosynthetic host in biotechnology

Engineering the interface between cellular chassis and synthetic biological systems

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references (p. 165-176).The aim of my thesis is to help enable the engineering of biological systems that behave in a predictable manner. Well-established techniques exist to engineer systems that behave as expected. Here, I apply such techniques to two aspects of the engineering of biological systems. First, I address the design and construction of standard biological devices in a manner that facilitates reuse in higher-order systems. I describe the design and construction of an exemplar device, an engineered cell-cell communication receiver using standard biological parts (refined genetic objects designed to support physical and functional composition). I adopt a conventional framework for describing the behavior of engineered devices and use the adopted framework to design and interpret experiments that describe the behavior of the receiver. The output of the device is the activity of a promoter reported in units of Polymerases Per Second (PoPS), a common signal carrier. Second, I begin to address the coupling that exists between engineered biological systems and the host cell, or chassis. I propose that the coupling between engineered biological systems and the cellular chassis might be reduced if fewer resources were shared between the system and the chassis. I describe the construction of cellular chassis expressing both T7 RNA polymerases (RNAP) and orthogonal ribosomes that are unused by the chassis but are available for use by an engineered system. I implement a network in which the orthogonal ribosomal RNA and the gene encoding T7 RNAP are transcribed by T7 RNAP. In turn, the orthogonal ribosomes translate the T7 RNAP message. In addition, the T7 RNAP and orthogonal ribosomes express a repressor that inhibits transcription of both the T7 RNAP and orthogonal ribosomes.(cont.) As a result, the orthogonal RNAP and ribosomes are auto-generating and self-regulating. The provision of resources unused by the cellular chassis and dedicated to an engineered biological system forms the beginnings of a biological virtual machine.by Bartholomew Canton.Ph.D

Development of novel orthogonal genetic circuits, based on extracytoplasmic function (ECF) σ factors

The synthetic biology field aims to apply the engineering 'design-build-test-learn' cycle for the implementation of synthetic genetic circuits modifying the behavior of biological systems. In order to reach this goal, synthetic biology projects use a set of fully characterized biological parts that subsequently are assembled into complex synthetic circuits following a rational, model-driven design. However, even though the bottom-up design approach represents an optimal starting point to assay the behavior of the synthetic circuits under defined conditions, the rational design of such circuits is often restricted by the limited number of available DNA building blocks. These usually consist only of a handful of transcriptional regulators that additionally are often borrowed from natural biological systems. This, in turn, can lead to cross-reactions between the synthetic circuit and the host cell and eventually to loss of the original circuit function. Thus, one of the challenges in synthetic biology is to design synthetic circuits that perform the designated functions with minor cross-reactions (orthogonality). To overcome the restrictions of the widely used transcriptional regulators, this project aims to apply extracytoplasmic function (ECF) σ factors in the design novel orthogonal synthetic circuits. ECFs are the smallest and simplest alternative σ factors that recognize highly specific promoters. ECFs represent one of the most important mechanisms of signal transduction in bacteria, indeed, their activity is often controlled by anti-σ factors. Even though it was shown that the overexpression of heterologous anti-σ factors can generate an adverse effect on cell growth, they represent an attractive solution to control ECF activity. Finally, to date, we know thousands of ECF σ factors, widespread among different bacterial phyla, that are identifiable together with the cognate promoters and anti-σ factors, using bioinformatic approaches. All the above-mentioned features make ECF σ factors optimal candidates as core orthogonal regulators for the design of novel synthetic circuits. In this project, in order to establish ECF σ factors as standard building blocks in the synthetic biology field, we first established a high throughput experimental setup. This relies on microplate reader experiments performed using a highly sensitive luminescent reporter system. Luminescent reporters have a superior signal-to-noise ratio when compared to fluorescent reporters since they do not suffer from the high auto-fluorescence background of the bacterial cell. However, they also have a drawback represented by the constant light emission that can generate undesired cross-talk between neighboring wells on a microplate. To overcome this limitation, we developed a computational algorithm that corrects for luminescence bleed-through and estimates the “true” luminescence activity for each well of a microplate. We show that the correcting algorithm preserves low-level signals close to the background and that it is universally applicable to different experimental conditions. In order to simplify the assembly of large ECF-based synthetic circuits, we designed an ECF toolbox in E. coli. The toolbox allows for the combinatorial assembly of circuits into expression vectors, using a library of reusable genetic parts. Moreover, it also offers the possibility of integrating the newly generated synthetic circuits into four different phage attachment (att) sites present in the genome of E. coli. This allows for a flawless transition between plasmid-encoded and chromosomally integrated genetic circuits, expanding the possible genetic configurations of a given synthetic construct. Moreover, our results demonstrate that the four att sites are orthogonal in terms of the gene expression levels of the synthetic circuits. With the purpose of rationally design ECF-based synthetic circuits and taking advantage of the ECF toolbox, we characterized the dynamic behavior of a set of 15 ECF σ factors, their cognate promoters, and relative anti-σs. Overall, we found that ECFs are non-toxic and functional and that they display different binding affinities for the cognate target promoters. Moreover, our results show that it is possible to optimize the output dynamic range of the ECF-based switches by changing the copy number of the ECFs and target promoters, thus, tuning the input/output signal ratio. Next, by combining up to three ECF-switches, we generated a set of “genetic-timer circuits”, the first synthetic circuits harboring more than one ECF. ECF-based timer circuits sequentially activate a series of target genes with increasing time delays, moreover, the behavior of the circuits can be predicted by a set of mathematical models. In order to improve the dynamic response of the ECF-based constructs, we introduced anti-σ factors in our synthetic circuits. By doing so we first confirmed that anti-σ factors can exert an adverse effect on the growth of E. coli, thus we explored possible solutions. Our results demonstrate that anti-σ factors toxicity can be partially alleviated by generating truncated, soluble variants of the anti-σ factors and, eventually, completely abolished via chromosomal integration of the anti-σ factor-based circuits. Finally, after demonstrating that anti-σ factors can be used to generate a tunable time delay among ECF expression and target promoter activation, we designed ECF/AS-suicide circuits. Such circuits allow for the time-delayed cell-death of E. coli and will serve as a prototype for the further development of ECF/AS-based lysis circuits