Recent advances and opportunities in synthetic logic gates engineering in living cells

Recently, a number of synthetic biologic gates including AND, OR, NOR, NOT, XOR and NAND have been engineered and characterized in a wide range of hosts. The hope in the emerging synthetic biology community is to construct an inventory of well-characterized parts and install distinct gene and circuit behaviours that are externally controllable. Though the field is still growing and major successes are yet to emerge, the payoffs are predicted to be significant. In this review, we highlight specific examples of logic gates engineering with applications towards fundamental understanding of network complexity and generating a novel socially useful applications.     

Considerations for using integral feedback control to construct a perfectly adapting synthetic gene network

It has long been known to control theorists and engineers that integral feedback control leads to, and is necessary for, “perfect” adaptation to step input perturbations in most systems. Consequently, implementation of this robust control strategy in a synthetic gene network is an attractive prospect. However, the nature of genetic regulatory networks (density-dependent kinetics and molecular signals that easily reach saturation) implies that the design and construction of such a device is not straightforward. In this study, we propose a generic two-promoter genetic regulatory network for the purpose of exhibiting perfect adaptation; our treatment highlights the challenges inherent in the implementation of a genetic integral controller. We also present a numerical case study for a specific realization of this two-promoter network, “constructed” using commonly available parts from the bacterium Escherichia coli. We illustrate the possibility of optimizing this network's transient response via analogy to a linear, free-damped harmonic oscillator. Finally, we discuss extensions of this two-promoter network to a proportional-integral controller and to a three-promoter network capable of perfect adaptation under conditions where first-order protein removal effects would otherwise disrupt the adaptation.     

Epistasis in a quantitative trait captured by a molecular model of transcription factor interactions

With technological advances in genetic mapping studies more of the genes and polymorphisms that underlie Quantitative Trait Loci (QTL) are now being identified. As the identities of these genes become known there is a growing need for an analysis framework that incorporates the molecular interactions affected by natural polymorphisms. As a step towards such a framework we present a molecular model of genetic variation in sporulation efficiency between natural isolates of the yeast, Saccharomyces cerevisiae. The model is based on the structure of the regulatory pathway that controls sporulation. The model captures the phenotypic variation between strains carrying different combinations of alleles at known QTL. Compared to a standard linear model the molecular model requires fewer free parameters, and has the advantage of generating quantitative hypotheses about the affinity of specific molecular interactions in different genetic backgrounds. Our analyses provide a concrete example of how the thermodynamic properties of protein-protein and protein-DNA interactions naturally give rise to epistasis, the non-linear relationship between genotype and phenotype. As more causative genes and polymorphisms underlying QTL are identified, thermodynamic analyses of quantitative traits may provide a useful framework for unraveling the complex relationship between genotype and phenotype.     

Cooperative assembly confers regulatory specificity and long-term genetic circuit stability

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Engineering the bilayer: Emerging genetic tool kits for mechanistic lipid biology

The structural diversity of lipids underpins the biophysical properties of cellular membranes, which vary across all scales of biological organization. Because lipid composition results from complex metabolic and transport pathways, its experimental control has been a major goal of mechanistic membrane biology. Here, we argue that in the wake of synthetic biology, similar metabolic engineering strategies can be applied to control the composition, physicochemical properties, and function of cell membranes. In one emerging area, titratable expression platforms allow for specific and genome-wide alterations in lipid biosynthetic genes, providing analog control over lipidome stoichiometry in membranes. Simultaneously, heterologous expression of biosynthetic genes and pathways has allowed for gain-of-function experiments with diverse lipids in non-native systems. Finally, we highlight future directions for tool development, including recently discovered lipid transport pathways to intracellular lipid pools. Further tool development providing synthetic control of membrane properties can allow biologists to untangle membrane lipid structure-associated functions.     

Dynamic metabolic engineering: New strategies for developing responsive cell factories

Metabolic engineering strategies have enabled improvements in yield and titer for a variety of valuable small molecules produced naturally in microorganisms, as well as those produced via heterologous pathways. Typically, the approaches have been focused on up‐ and downregulation of genes to redistribute steady‐state pathway fluxes, but more recently a number of groups have developed strategies for dynamic regulation, which allows rebalancing of fluxes according to changing conditions in the cell or the fermentation medium. This review highlights some of the recently published work related to dynamic metabolic engineering strategies and explores how advances in high‐throughput screening and synthetic biology can support development of new dynamic systems. Dynamic gene expression profiles allow trade‐offs between growth and production to be better managed and can help avoid build‐up of undesired intermediates. The implementation is more complex relative to static control, but advances in screening techniques and DNA synthesis will continue to drive innovation in this field.     

A simple way for sequential assignment in isotopically enriched proteins using a H(N)CACO correlation

Summary A simplified scheme for sequential assignment in isotopically enriched proteins is presented. It is based on the standard triple resonance experiments HNCO, HN(CO)CA, HNCA and a modified H(N)CACO correlation, in which both of the H^N connectivities to the CA/C pair of residue i and i-1 are observed. The H(N)CACO was tested on uniformly ¹³C/¹⁵N enriched P13 domain of mannose permease (31 kDa).     

Beyond directed evolution: Darwinian selection as a tool for synthetic biology

Synthetic biology is an engineering approach that seeks to design and construct new biological parts, devices and systems, as well as to re-design existing components. However, rationally designed synthetic circuits may not work as expected due to the context-dependence of biological parts. Darwinian selection, the main mechanism through which evolution works, is a major force in creating biodiversity and may be a powerful tool for synthetic biology. This article reviews selection-based techniques and proposes strict Darwinian selection as an alternative approach for the identification and characterization of parts. Additionally, a strategy for fine-tuning of relatively complex circuits by coupling them to a master standard circuit is discussed.     

Regulation by competition: a hidden layer of gene regulatory network

Background: Molecular competition brings about trade-offs of shared limited resources among the cellular components, and thus introduces a hidden layer of regulatory mechanism by connecting components even without direct physical interactions. Several molecular competition scenarios have been observed recently, but there is still a lack of systematic quantitative understanding to reveal the essence of molecular competition. Methods: Here, by abstracting the analogous competition mechanism behind diverse molecular systems, we built a unified coarse-grained competition motif model to systematically integrate experimental evidences in these processes and analyzed general properties shared behind them from steady-state behavior to dynamic responses. Results: We could predict in what molecular environments competition would reveal threshold behavior or display a negative linear dependence. We quantified how competition can shape regulator-target dose-response curve, modulate dynamic response speed, control target expression noise, and introduce correlated fluctuations between targets. Conclusions: This work uncovered the complexity and generality of molecular competition effect as a hidden layer of gene regulatory network, and therefore provided a unified insight and a theoretical framework to understand and employ competition in both natural and synthetic systems.     

Corona Detective: a simple,scalable, and robust SARS-CoV-2 detection method based on reverse transcription loop-mediated isothermal amplification

Surveillance screening at scale to identify people infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prior to extensive transmission is key to bringing an end to the coronavirus disease 2019 (COVID-19) pandemic, even though vaccinations have already begun. Here we describe Corona Detective, a sensitive and rapid molecular test to detect the virus, based on loop-mediated isothermal amplification, which could be applied anywhere at low cost. Critically, the method uses freeze-dried reagents, readily shipped without cold-chain dependence. The reaction detects the viral nucleocapsid gene through a sequence-specific quenched-fluorescence readout, which avoids false positives and also allows multiplex detection with an internal control cellular RNA. Corona Detective can be used in 8-tube strips to be read with a simple open-design fluorescence detector. Other methods to use and produce Corona Detective locally in a variety of formats are possible and already openly shared. Detection specificity is ensured through inclusion of positive and negative control reactions to run in parallel with the diagnostic reactions. A simple user protocol, including sample preparation, and a bioinformatics pipeline to ensure that viral variants will still be detectable with SARS-CoV-2 primer sets complete the method. Through rapid production and distribution of Corona Detective reactions, quite inexpensive at scale, daily or weekly surveillance testing of large populations, without waiting for symptoms to develop, is anticipated, in combination with vaccination campaigns, to finally control this pandemic.     

Question 7: Construction of a Semi-Synthetic Minimal Cell: A Model for Early Living Cells

Using a Synthetic Biology approach we are building a semi-synthetic minimal cell. This represents an exercise to shape a minimal-cell model system recalling the simplicity of early living cells in early evolution. We have recently introduced into liposome compartments a minimal set of enzymes named “Puresystem” (PS) synthesizing EGFP proteins. To establish reproduction of the shell compartment with a minimal set of genes we have cloned the genes for the Fatty Acid Synthase (FAS) type I enzymes. These FAS genes introduced into liposomes, translated into FAS enzymes by PS and in the presence of precursors produce fatty acids. The resulting release of fatty acid molecules within liposome vesicles should promote vesicle growth and reproduction. The core reproduction of a minimal cell corresponding to the replication of the minimal genome will require a few genes for the DNA replication and the PS, and a minimum set of genes for the synthesis of t-RNAs. In future the reconstruction of a minimal ribosome will bring the number of genes for ribosomal proteins from 54 of an existing minimal genome down to 30–20 genes. A Synthetic Biology approach could bring the number of essential genes for a minimal cell down to 100 or less. International School of Complexity–4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.