Synthetic Biological Devices Research Articles

Variable orthogonality of serine integrase interactions within the ϕC31 family

Serine integrases are phage- (or mobile element-) encoded enzymes that catalyse site-specific recombination reactions between a short DNA sequence on the phage genome (attP) and a corresponding host genome sequence (attB), thereby integrating the phage DNA into the host genome. Each integrase has its unique pair of attP and attB sites, a feature that allows them to be used as orthogonal tools for genome modification applications. In the presence of a second protein, the Recombination Directionality Factor (RDF), integrase catalyses the reverse excisive reaction, generating new recombination sites, attR and attL. In addition to promoting attR x attL reaction, the RDF inhibits attP x attB recombination. This feature makes the directionality of integrase reactions programmable, allowing them to be useful for building synthetic biology devices. In this report, we describe the degree of orthogonality of both integrative and excisive reactions for three related integrases (ϕC31, ϕBT1, and TG1) and their RDFs. Among these, TG1 integrase is the most active, showing near complete recombination in both attP x attB and attR x attL reactions, and the most directional in the presence of its RDF. Our findings show that there is varying orthogonality among these three integrases – RDF pairs. ϕC31 integrase was the least selective, with all three RDFs activating it for attR x attL recombination. Similarly, ϕC31 RDF was the least effective among the three RDFs in promoting the excisive activities of the integrases, including its cognate ϕC31 integrase. ϕBT1 and TG1 RDFs were noticeably more effective than ϕC31 RDF at inhibiting attP x attB recombination by their respective integrases, making them more suitable for building reversible genetic switches. AlphaFold-Multimer predicts very similar structural interactions between each cognate integrase – RDF pair. The binding surface on the RDF is much more conserved than the binding surface on the integrase, an indication that specificity is determined more by the integrase than the RDF. Overall, the observed weak integrase/RDF orthogonality across the three enzymes emphasizes the need for identifying and characterizing more integrase – RDF pairs. Additionally, the ability of a particular integrase’s preferred reaction direction to be controlled to varying degrees by non-cognate RDFs provides a path to tunable, non-binary genetic switches.

Analysis of heat, mass transfer, and microorganism density rates in bioconvection nanofluid flow under the influence of thermal radiation using response surface methodology

This article presents innovative studies on bioconvection caused by microorganisms in a nanofluid flow with nonlinear radiation effects over a vertical plate. The set of governing partial differential equations is transformed by the similarity transformation into a set of nonlinearly coupled ordinary differential equations (ODEs). These are then solved numerically using the fourth-order Runge–Kutta method. The current findings are in remarkable agreement with the previous studies found in the literature. It is noted that the impact of bioconvection Lewis number and Peclet number on the microbe density rate profile gets reduced, and the increasing effect of the nonlinear thermal radiation parameter on the temperature profile is observed, which maintains the heat transmission distances inside the boundary layer. Additionally, the interactions of heat, mass transfer, and microbe density rates with the selected input parameters are discussed using the response surface methodology (RSM). This model is more applicable in a variety of fields, including synthetic biology, biofuel cells, and drug delivery devices.

Noncanonical Streptococcus sanguinis ComCDE circuitry integrates environmental cues in transformation outcome decision

Natural competence is the principal driver of streptococcal evolution. While acquisition of new traits could facilitate rapid fitness improvement for bacteria, entry into the competent state is a highly orchestrated event, involving an interplay between various pathways. We present a new type of competence-predation coordination mechanism in Streptococcus sanguinis. Unlike other streptococci that mediate competence through the ComABCDE regulon, several key components are missing in the S.sanguinis ComCDE circuitry. We assembled two synthetic biology devices linking competence-stimulating peptide (CSP) cleavage and export with a quantifiable readout to unravel the unique features of the S.sanguinis circuitry. Our results revealed the ComC precursor cleavage pattern and the two host ABC transporters implicated in the export of the S.sanguinis CSP. Moreover, we discovered a ComCDE-dependent bacteriocin locus. Overall, this study presents a mechanism for commensal streptococci to maximize transformation outcome in a fluid environment through extensive circuitry rewiring.

Cellular Computational Logic Using Toehold Switches.

The development of computational logic that carries programmable and predictable features is one of the key requirements for next-generation synthetic biological devices. Despite considerable progress, the construction of synthetic biological arithmetic logic units presents numerous challenges. In this paper, utilizing the unique advantages of RNA molecules in building complex logic circuits in the cellular environment, we demonstrate the RNA-only bitwise logical operation of XOR gates and basic arithmetic operations, including a half adder, a half subtractor, and a Feynman gate, in Escherichia coli. Specifically, de-novo-designed riboregulators, known as toehold switches, were concatenated to enhance the functionality of an OR gate, and a previously utilized antisense RNA strategy was further optimized to construct orthogonal NIMPLY gates. These optimized synthetic logic gates were able to be seamlessly integrated to achieve final arithmetic operations on small molecule inputs in cells. Toehold-switch-based ribocomputing devices may provide a fundamental basis for synthetic RNA-based arithmetic logic units or higher-order systems in cells.

Identification and characterization of L- and D-lactate-inducible systems from Escherichia coli MG1655, Cupriavidus necator H16 and Pseudomonas species

Lactic acid is an important platform chemical used for the production of various compounds including polylactic acid (PLA). Optically pure L- and D-lactic acids are required to obtain high quality PLA. To advance the development and selection of microbial strains for improved production of lactic acid enantiomers, a high-throughput screening, dynamic pathway control, or real-time monitoring are often applied. Inducible gene expression systems and their application in the genetically encoded biosensors contribute to the development of these techniques and are important devices for the advancement of lactic acid biotechnology. Here, we identify and characterize eleven lactate-inducible systems from Escherichia coli, Cupriavidus necator, and Pseudomonas spp. The specificity and dynamics of these systems in response to L- and D-lactate, or structurally similar compounds are investigated. We demonstrate that the inducible systems EcLldR/PlldP and CnGntR/PH16_RS19190 respond only to the L-lactate, exhibiting approximately 19- and 24-fold induction, respectively. Despite neither of the examined bacteria possess the D-lactate-specific inducible system, the PaPdhR/PlldP and PfPdhR/PlldP are induced approximately 37- and 366-fold, respectively, by D-lactate and can be used for developing biosensor with improved specificity. The findings of this study provide an insight into understanding of L- and D-lactate-inducible systems that can be employed as sensing and tuneable devices in synthetic biology.

Detection of pks Island mRNAs Using Toehold Sensors in Escherichia coli.

Synthetic biologists have applied biomolecular engineering approaches toward the goal of novel biological devices and have shown progress in diverse areas of medicine and biotechnology. Especially promising is the application of synthetic biological devices towards a novel class of molecular diagnostics. As an example, a de-novo-designed riboregulator called toehold switch, with its programmability and compatibility with field-deployable devices showed promising in vitro applications for viral RNA detection such as Zika and Corona viruses. However, the in vivo application of high-performance RNA sensors remains challenging due to the secondary structure of long mRNA species. Here, we introduced ‘Helper RNAs’ that can enhance the functionality of toehold switch sensors by mitigating the effect of secondary structures around a target site. By employing the helper RNAs, previously reported mCherry mRNA sensor showed improved fold-changes in vivo. To further generalize the Helper RNA approaches, we employed automatic design pipeline for toehold sensors that target the essential genes within the pks island, an important target of biomedical research in connection with colorectal cancer. The toehold switch sensors showed fold-changes upon the expression of full-length mRNAs that apparently depended sensitively on the identity of the gene as well as the predicted local structure within the target region of the mRNA. Still, the helper RNAs could improve the performance of toehold switch sensors in many instances, with up to 10-fold improvement over no helper cases. These results suggest that the helper RNA approaches can further assist the design of functional RNA devices in vivo with the aid of the streamlined automatic design software developed here. Further, our solutions for screening and stabilizing single-stranded region of mRNA may find use in other in vivo mRNA-sensing applications such as cas13 crRNA design, transcriptome engineering, and trans-cleaving ribozymes.

Access and benefit‐sharing DNA Componentry for plant synthetic biology: Bioparts expressed in plant chassis

Societal Impact StatementThe “Parts Agenda” is an approach to synthetic biology that fragments genetic resources into functional bioparts to help design and build biological devices and systems. Access and benefit‐sharing (ABS), and the issue of how to regulate digital sequence information (DSI) within the current ABS regime, poses a problem for synthetic biology because it assumes fragmented and abstracted bioparts can be traced to their country of origin for the purposes of benefit‐sharing, and that contributions to information and knowledge can be quantified and appropriately valued. Any DSI regulatory solutions should account for genetic resource fragmentation and other complexities of modern scientific practice.Summary The inclusion of digital sequence information (DSI, including genetic sequence data) in the existing access and benefit‐sharing (ABS) regime will alter the practice of synthetic biology. The potential impediments could be magnified for the “Parts Agenda”: the approach to synthetic biology that fragments genetic resources into their smallest functional units to create standardized, interchangeable “bioparts”, the building blocks for assembling synthetic biological devices. These biological devices are themselves interchangeable and can be used to engineer higher order synthetic biological systems. This article examines how the extension of ABS laws to include DSI could foreseeably apply to the creation and use of plant‐derived and other bioparts in engineered biological devices expressed in plant chassis. The article demonstrates that ABS issues will be similar for all approaches to synthetic biology, but that the Parts Agenda is uniquely exposed to the potential regulatory burden of bilateral ABS transactions between users and providers of genetic resources. The original vision for bioparts was one of openness and sharing, with access and use unencumbered by intellectual property. This article shows that open access to bioparts will not last long if DSI is enclosed within the current ABS regime, destabilizing the values of openness and sharing in synthetic biology that are ostensibly foundational to this still developing field.

A Standardized Inverter Package Borne by Broad Host Range Plasmids for Genetic Circuit Design in Gram-Negative Bacteria

Genetically encoded logic gates, especially inverters-NOT gates-are the building blocks for designing circuits, engineering biosensors, or decision-making devices in synthetic biology. However, the repertoire of inverters readily available for different species is rather limited. In this work, a large whole of NOT gates that was shown to function previously in a specific strain of Escherichia coli, was recreated as broad host range (BHR) collection of constructs assembled in low, medium, and high copy number plasmid backbones of the SEVA (Standard European Vector Architecture) collection. The input/output function of each of the gates was characterized and parametrized in the environmental bacterium and metabolic engineering chassis Pseudomonas putida. Comparisons of the resulting fluorescence cytometry data with those published for the same gates in Escherichia coli provided useful hints on the portability of the corresponding gates. The hereby described inverter package (20 different versions of 12 distinct gates) borne by BHR plasmids thus becomes a toolbox of choice for designing genetic circuitries in a variety of Gram-negative species other than E. coli.

Transforming yeast peroxisomes into microfactories for the efficient production of high-value isoprenoids

Current approaches for the production of high-value compounds in microorganisms mostly use the cytosol as a general reaction vessel. However, competing pathways and metabolic cross-talk frequently prevent efficient synthesis of target compounds in the cytosol. Eukaryotic cells control the complexity of their metabolism by harnessing organelles to insulate biochemical pathways. Inspired by this concept, herein we transform yeast peroxisomes into microfactories for geranyl diphosphate-derived compounds, focusing on monoterpenoids, monoterpene indole alkaloids, and cannabinoids. We introduce a complete mevalonate pathway in the peroxisome to convert acetyl-CoA to several commercially important monoterpenes and achieve up to 125-fold increase over cytosolic production. Furthermore, peroxisomal production improves subsequent decoration by cytochrome P450s, supporting efficient conversion of (S)-(-)-limonene to the menthol precursor trans-isopiperitenol. We also establish synthesis of 8-hydroxygeraniol, the precursor of monoterpene indole alkaloids, and cannabigerolic acid, the cannabinoid precursor. Our findings establish peroxisomal engineering as an efficient strategy for the production of isoprenoids.

High-Throughput Analysis and Engineering of Ribozymes and Deoxyribozymes by Sequencing.

Ribozymes and deoxyribozymes are catalytic RNA and DNA, respectively, that catalyze chemical reactions such as self-cleavage or ligation reactions. While some ribozymes are found in nature, a larger variety of ribozymes and deoxyribozymes have been discovered by in vitro selection from random sequences. These catalytic nucleic acids, especially ribozymes, are of fundamental interest because they are crucial for the RNA world hypothesis, which suggests that RNA played a central role in both the propagation of genetic information and catalyzing metabolic reactions in primordial life prior to the emergence of proteins and DNA. On the practical side, catalytic nucleic acids have been extensively engineered for various applications, such as biosensors and genetic devices for synthetic biology. Therefore, it is important to gain a deeper understanding of the sequence-function relationships of ribozymes and deoxyribozymes.Mutational analysis, or measurements of activities of catalytic nucleic acid mutants, is one of the most fundamental approaches for that purpose. Mutations that abolish, reduce, retain, or even increase activity provide useful information about nucleic acid catalysts for engineering and other purposes. However, methods for mutational analysis of ribozymes and deoxyribozymes have not evolved much for decades, requiring tedious and low-throughput assays (e.g., gel electrophoresis) of individually prepared mutants. This has prevented researchers from performing quantitative mutational analysis of ribozymes and deoxyribozymes on a large scale.To address this limitation, we developed a massively parallel ribozyme and deoxyribozyme assay strategy that allows >104 assays using high-throughput sequencing (HTS). We used HTS to literally count the number of cleaved (or ligated) and uncleaved (or unligated) ribozyme (or deoxyribozyme) sequences and calculated the activities of each mutant in a reaction mixture. This simple yet powerful strategy was applied to analyze the mutational effects of various natural and synthetic ribozymes and deoxyribozymes at scales impossible for conventional mutational analysis. These large-scale sequence-function data sets were used to better understand the functional consequences of mutations and to engineer ribozymes for practical applications. Furthermore, these newly available data are motivating researchers to employ more rigorous computational methods to extract additional insights such as structural information and nonlinear effects of multiple mutations. The new HTS-based assay strategy is distinct from and complementary to a related strategy that uses HTS to analyze ribozyme and deoxyribozyme populations subjected to in vitro selection. Postselection sequencing can cover a larger sequence space, although it does not directly quantify the activities of ribozyme and deoxyribozyme mutants. With further advances in DNA sequencing technologies and computational methods, there should be more opportunities to harness the power of HTS to deepen our understanding of catalytic nucleic acids and enhance our ability to engineer them for even more applications.

Regulation of Electromagnetic Perceptive Gene Using Ferromagnetic Particles for the External Control of Calcium Ion Transport.

Developing synthetic biological devices to allow the noninvasive control of cell fate and function, in vivo can potentially revolutionize the field of regenerative medicine. To address this unmet need, we designed an artificial biological “switch” that consists of two parts: (1) the electromagnetic perceptive gene (EPG) and (2) magnetic particles. Our group has recently cloned the EPG from the Kryptopterus bicirrhis (glass catfish). The EPG gene encodes a putative membrane-associated protein that responds to electromagnetic fields (EMFs). This gene’s primary mechanism of action is to raise the intracellular calcium levels or change in flux through EMF stimulation. Here, we developed a system for the remote regulation of [Ca2+]i (i.e., intracellular calcium ion concentration) using streptavidin-coated ferromagnetic particles (FMPs) under a magnetic field. The results demonstrated that the EPG-FMPs can be used as a molecular calcium switch to express target proteins. This technology has the potential for controlled gene expression, drug delivery, and drug developments.

Manipulation of Bacterial Signaling Using Engineered Histidine Kinases.

Two-component systems allow bacteria to respond to changes in environmental or cytosolic conditions through autophosphorylation of a histidine kinase (HK) and subsequent transfer of the phosphate group to its downstream cognate response regulator (RR). The RR then elicits a cellular response, commonly through regulation of transcription. Engineering two-component system signaling networks provides a strategy to study bacterial signaling mechanisms related to bacterial cell survival, symbiosis, and virulence, and to develop sensory devices in synthetic biology. Here we focus on the principles for engineering the HK to identify unknown signal inputs, test signal transmission mechanisms, design small molecule sensors, and rewire two-component signaling networks.

TASBE Flow Analytics: A Package for Calibrated Flow Cytometry Analysis.

Flow cytometry is a powerful method for high-throughput precision measurement of cell fluorescence and size. Effective use of this tool for quantification of synthetic biology devices and circuits, however, generally requires careful application of complex multistage workflows for calibration, filtering, and analysis with appropriate statistics. The TASBE Flow Analytics package provides a free, open, and accessible implementation of such workflows in a form designed for high-throughput analysis of large synthetic biology data sets. Given a set of experimental samples and controls, this package can process them to output calibrated data, quantitative analyses and comparisons, automatically generated figures, and detailed debugging and diagnostic reports in both human-readable and machine-readable forms. TASBE Flow Analytics can be used through a simple user-friendly interactive Excel interface, as a library supporting Matlab, Octave, or Python interactive sessions, or as a component integrated into automated workflows.

Applications and limitations of regulatory RNA elements in synthetic biology and biotechnology.

Synthetic biology requires the design and implementation of novel enzymes, genetic circuits or even entire cells, which can be controlled by the user. RNA-based regulatory elements have many important functional properties in this regard, such as their modular nature and their ability to respond to specific external stimuli. These properties have led to the widespread exploration of their use as gene regulation devices in synthetic biology. In this review, we focus on two major types of RNA elements: riboswitches and RNA thermometers (RNATs). We describe their general structure and function, before discussing their potential uses in synthetic biology (e.g. in the production of biofuels and biodegradable plastics). We also discuss their limitations, and novel strategies to implement RNA-based regulatory devices in biotechnological applications. We close with a description of some common model organisms used in synthetic biology, with a focus on the current applications and limitations of RNA-based regulation.

Directed Assembly of Homopentameric Cholera Toxin B-Subunit Proteins into Higher-Order Structures Using Coiled-Coil Appendages.

The self-assembly of proteins into higher order structures is ubiquitous in living systems. It is also an essential process for the bottom-up creation of novel molecular architectures and devices for synthetic biology. However, the complexity of protein–protein interaction surfaces makes it challenging to mimic natural assembly processes in artificial systems. Indeed, many successful computationally designed protein assemblies are prescreened for “designability”, limiting the choice of components. Here, we report a simple and pragmatic strategy to assemble chosen multisubunit proteins into more complex structures. A coiled-coil domain appended to one face of the pentameric cholera toxin B-subunit (CTB) enabled the ordered assembly of tubular supra-molecular complexes. Analysis of a tubular structure determined by X-ray crystallography has revealed a hierarchical assembly process that displays features reminiscent of the polymorphic assembly of polyomavirus proteins. The approach provides a simple and straightforward method to direct the assembly of protein building blocks which present either termini on a single face of an oligomer. This scaffolding approach can be used to generate bespoke supramolecular assemblies of functional proteins. Additionally, structural resolution of the scaffolded assemblies highlight “native-state” forced protein–protein interfaces, which may prove useful as starting conformations for future computational design.

Rationalizing Context-Dependent Performance of Dynamic RNA Regulatory Devices.

The ability of RNA to sense, regulate, and store information is an attractive attribute for a variety of functional applications including the development of regulatory control devices for synthetic biology. RNA folding and function is known to be highly context sensitive, which limits the modularity and reuse of RNA regulatory devices to control different heterologous sequences and genes. We explored the cause and effect of sequence context sensitivity for translational ON riboswitches located in the 5' UTR, by constructing and screening a library of N-terminal synonymous codon variants. By altering the N-terminal codon usage we were able to obtain RNA devices with a broad range of functional performance properties (ON, OFF, fold-change). Linear regression and calculated metrics were used to rationalize the major determining features leading to optimal riboswitch performance, and to identify multiple interactions between the explanatory metrics. Finally, partial least squared (PLS) analysis was employed in order to understand the metrics and their respective effect on performance. This PLS model was shown to provide good explanation of our library. This study provides a novel multivariant analysis framework to rationalize the codon context performance of allosteric RNA-devices. The framework will also serve as a platform for future riboswitch context engineering endeavors.

Synthetic Whole-Cell Biodevices for Targeted Degradation of Antibiotics

Synthetic biology enables infinite possibilities in biotechnology via employing genetic modules. However, not many researches have explored the potentials of synthetic biology in environmental bioprocesses. In this study, we introduced a genetic module harboring the codon-optimized tetracycline degrading gene, tetX.co, into the model host, Escherichia coli, and generated a prototypal whole-cell biodevice for the degradation of a target antibiotic. Our results suggested that E. coli with the tetX.co-module driven by either the PJ23119 or PBAD promoters conferred resistance up to 50 μg/mL of tetracycline and degrades over 95% of tetracycline within 24 h. The detoxification ability of tetX was further verified in conditioned media by typical E. coli K-12 and B strains as well as Shewanella oneidensis. Our strategy demonstrated the feasibility of introducing genetic modules into model hosts to enable environmental functions, and this work will inspire more environmental innovations through synthetic biological devices.

Extended Metabolic Space Modeling.

Determining the fraction of the chemical space that can be processed in vivo by using natural and synthetic biology devices is crucial for the development of advanced synthetic biology applications. The extended metabolic space is a coding system based on molecular signatures that enables the derivation of reaction rules for metabolic reactions and the enumeration of all possible substrates and products corresponding to the rules. The extended metabolic space expands capabilities for controlling the production, processing, sensing, and the release of specific molecules in chassis organisms.

Synthetic self‐patterning and morphogenesis in mammalian cells: a proof‐of‐concept step towards synthetic tissue development

This study reports a proof-of-concept study as a step toward synthetic-biological morphogenesis of tissues. Events in normal animal development usually follow the sequence: patterning → differential gene expression → morphogenesis. A synthetic biological approach to development might follow a similar sequence, with each stage under the control of synthetic biological modules. The authors have constructed and published a synthetic module that drives self-organised patterning of mammalian cell populations into patches of different cell types. Here, as a proof of concept, they extend the self-patterning module with a morphogenetic effector that drives elective cell death in just one cell type. The result is a self-constructing pattern of two cell types, one of which can be selectively eliminated to leave remaining cells as a monolayer with a net-like structure. This simple device demonstrates and validates the idea of coupling synthetic biological morphogenetic effectors to synthetic biological patterning devices. It opens the path to engineering more sophisticated structures and, perhaps eventually, tissues.

Feedback Control and Synthetic Biology: Constraints on Design

Synthetic Biology is an emerging field at the interface of biology and engineering, concerned with the design and implementation of synthetic biological parts, devices and systems. With applications ranging from industrial biosynthesis of chemicals to treatment and prevention of disease, Synthetic Biology holds great promise, but faces several challenges due to the uncertainties and noise inherent in biological systems. In this paper we review recent progress in the design and testing of biological control systems that aim to overcome these limitations. We then use classical control theory to derive a number of design constraints for implementation of linear control systems that achieve adaptation and disturbance rejection. Finally, we design a linear system for rejection of ramp-type disturbances, and from this demonstrate how the derived linear system constraints can be embedded in a more realistic non-linear biological context.