Contextualizing context for synthetic biology--identifying causes of failure of synthetic biological systems.

Despite the efforts that bioengineers have exerted in designing and constructing biological processes that function according to a predetermined set of rules, their operation remains fundamentally circumstantial. The contextual situation in which molecules and single-celled or multi-cellular organisms find themselves shapes the way they interact, respond to the environment and process external information. Since the birth of the field, synthetic biologists have had to grapple with contextual issues, particularly when the molecular and genetic devices inexplicably fail to function as designed when tested in vivo. In this review, we set out to identify and classify the sources of the unexpected divergences between design and actual function of synthetic systems and analyze possible methodologies aimed at controlling, if not preventing, unwanted contextual issues

Developments in the tools and methodologies of synthetic biology.

Synthetic biology is principally concerned with the rational design and engineering of biologically based parts, devices, or systems. However, biological systems are generally complex and unpredictable, and are therefore, intrinsically difficult to engineer. In order to address these fundamental challenges, synthetic biology is aiming to unify a body of knowledge from several foundational scientific fields, within the context of a set of engineering principles. This shift in perspective is enabling synthetic biologists to address complexity, such that robust biological systems can be designed, assembled, and tested as part of a biological design cycle. The design cycle takes a forward-design approach in which a biological system is specified, modeled, analyzed, assembled, and its functionality tested. At each stage of the design cycle, an expanding repertoire of tools is being developed. In this review, we highlight several of these tools in terms of their applications and benefits to the synthetic biology community

Synthetic Gene Circuits: Design with Directed Evolution

Synthetic circuits offer great promise for generating insights into nature's underlying design principles or forward engineering novel biotechnology applications. However, construction of these circuits is not straightforward. Synthetic circuits generally consist of components optimized to function in their natural context, not in the context of the synthetic circuit. Combining mathematical modeling with directed evolution offers one promising means for addressing this problem. Modeling identifies mutational targets and limits the evolutionary search space for directed evolution, which alters circuit performance without the need for detailed biophysical information. This review examines strategies for integrating modeling and directed evolution and discusses the utility and limitations of available methods

Dynamic control of endogenous metabolism with combinatorial logic circuits

Controlling gene expression during a bioprocess enables real-time metabolic control, coordinated cellular responses, and staging order-of-operations. Achieving this with small molecule inducers is impractical at scale and dynamic circuits are difficult to design. Here, we show that the same set of sensors can be integrated by different combinatorial logic circuits to vary when genes are turned on and off during growth. Three Escherichia coli sensors that respond to the consumption of feedstock (glucose), dissolved oxygen, and by-product accumulation (acetate) are constructed and optimized. By integrating these sensors, logic circuits implement temporal control over an 18-h period. The circuit outputs are used to regulate endogenous enzymes at the transcriptional and post-translational level using CRISPRi and targeted proteolysis, respectively. As a demonstration, two circuits are designed to control acetate production by matching their dynamics to when endogenous genes are expressed (pta or poxB) and respond by turning off the corresponding gene. This work demonstrates how simple circuits can be implemented to enable customizable dynamic gene regulation.Synthetic Biology Engineering Research Center (SynBERC EEC0540879)United States. Office of Naval Research. Multidisciplinary University Research Initiative (N00014‐13‐1‐0074)United States. Department of Energy (DE‐SC0018368

Gene Regulatory Compatibility in Bacteria: Consequences for Synthetic Biology and Evolution

Mechanistic understanding of gene regulation is crucial for rational engineering of new genetic systems through synthetic biology. Genetic engineering efforts in new organisms are often hampered by a lack of knowledge about how regulatory components function in new host contexts. This dissertation focuses on efforts to overcome these challenges through the development of generalizable experimental methods for studying the behavior of DNA regulatory sequences in diverse species at large-scale. Chapter 2 describes experimental approaches for quantitatively assessing the functions of thousands of diverse natural regulatory sequences through a combination of metagenomic mining, high-throughput DNA synthesis and deep sequencing. By employing these methods in three distinct bacterial species, we revealed striking functional differences in gene regulatory capacity. We identified regulatory sequences with activity levels with activity levels spanning several orders of magnitude, which will aid in efforts to engineer diverse bacterial species. We also demonstrate functional species-selective gene circuits with programmable host behaviors that may be useful for microbial community engineering. In Chapter 3 we provide evidence for the evolution of altered stringency in σ70-mediated transcriptional activation based on patterns of initiation and activity from promoters of diverse compositions. We show that the contrast in GC content between a regulatory element and the host genome dictates both the likelihood and the magnitude of expression. We also discuss the potential implications of this proposed mechanism on horizontal gene transfer. The next two chapters focus on efforts aimed at extending the high-throughput methods described in earlier chapters to new organisms. Chapter 4 presents an in vitro approach for multiplexed gene expression profiling. Through the development and use of cell-free expression systems made from diverse bacteria, it was possible to rapidly acquire thousands of transcriptional measurements in small volume reactions, enabling functional comparisons of regulatory sequence function across multiple species. In Chapter 5 we characterize the restriction-modification system repertoires of several commensal bacterial species. We also describe ongoing efforts to develop methods for bypassing these systems in order to increase transformation efficiencies in species that are difficult or impossible to transform using current approaches

Measuring and modeling energy and power consumption in living microbial cells with a synthetic ATP reporter

Background: Adenosine triphosphate (ATP) is the main energy carrier in living organisms, critical for metabolism and essential physiological processes. In humans, abnormal regulation of energy levels (ATP concentration) and power consumption (ATP consumption flux) in cells is associated with numerous diseases from cancer, to viral infection and immune dysfunction, while in microbes it influences their responses to drugs and other stresses. The measurement and modeling of ATP dynamics in cells is therefore a critical component in understanding fundamental physiology and its role in pathology. Despite the importance of ATP, our current understanding of energy dynamics and homeostasis in living cells has been limited by the lack of easy-to-use ATP sensors and the lack of models that enable accurate estimates of energy and power consumption related to these ATP dynamics. Here we describe a dynamic model and an ATP reporter that tracks ATP in E. coli over different growth phases. Results: The reporter is made by fusing an ATP-sensing rrnB P1 promoter with a fast-folding and fast-degrading GFP. Good correlations between reporter GFP and cellular ATP were obtained in E. coli growing in both minimal and rich media and in various strains. The ATP reporter can reliably monitor bacterial ATP dynamics in response to nutrient availability. Fitting the dynamics of experimental data corresponding to cell growth, glucose, acetate, dissolved oxygen, and ATP yielded a mathematical and circuit model. This model can accurately predict cellular energy and power consumption under various conditions. We found that cellular power consumption varies significantly from approximately 0.8 and 0.2 million ATP/s for a tested strain during lag and stationary phases to 6.4 million ATP/s during exponential phase, indicating ~ 8–30-fold changes of metabolic rates among different growth phases. Bacteria turn over their cellular ATP pool a few times per second during the exponential phase and slow this rate by ~ 2–5-fold in lag and stationary phases. Conclusion: Our rrnB P1-GFP reporter and kinetic circuit model provide a fast and simple way to monitor and predict energy and power consumption dynamics in bacterial cells, which can impact fundamental scientific studies and applied medical treatments in the future

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

Promoter engineering to optimise recombinant periplasmic Fab' fragment production in Escherichia coli

Fab' fragments have become an established class of biotherapeutic over the last two decades. Likewise, developments in synthetic biology are providing ever more powerful techniques for designing bacterial genes, gene networks and entire genomes that can be used to the improve industrial performance of cells used for production of biotherapeutics. We have previously observed significant leakage of an exogenous therapeutic Fab' fragment into the growth medium during high cell density cultivation of an Escherichia coli production strain. In this study we sought to apply a promoter engineering strategy to address the issue of Fab' fragment leakage and its consequent bioprocess challenges. We used site directed mutagenesis to convert the Ptac promoter, present in the plasmid, pTTOD-A33 Fab', to a Ptic promoter which has been shown by others to direct expression at a 35% reduced rate compared to Ptac . We characterised the resultant production trains in which either Ptic or Ptac promoters direct Fab' fragment expression. The Ptic promoter strain showed a 25-30% reduction in Fab' expression relative to the original Ptac strain. Reduced Fab' leakage and increased viability over the course of a fed-batch fermentation were also observed for the Ptic promoter strain. We conclude that cell design steps such as the Ptac to Ptic promoter conversion reported here, can yield significant process benefit and understanding with respect to periplasmic Fab' fragment production. It remains an open question as to whether the influence of transgene expression on periplasmic retention is mediated by global metabolic burden effects or periplasm overcapacity

The Effect of Compositional Context on Synthetic Gene Networks

It is well known that synthetic gene expression is highly sensitive to how genetic elements (promoter structure, spacing regions between promoter and coding sequences, ribosome binding sites, etc.) are spatially configured. An important topic that has received far less attention is how the compositional context, or spatial arrangement, of entire genes within a synthetic gene network affects their individual expression levels. In this paper we show, both quantitatively and qualitatively, that compositional context significantly alters transcription levels in synthetic gene networks. We demonstrate that key characteristics of gene induction, such as ultra-sensitivity and dynamic range, strongly depend on compositional context. We postulate that supercoiling can be used to explain this interference and validate this hypothesis through modeling and a series of in vitro supercoiling relaxation experiments. This compositional interference enables a novel form of feedback in synthetic gene networks. We illustrate the use of this feedback by redesigning the toggle switch to incorporate compositional context. We show the context-optimized toggle switch has improved threshold detection and memory properties