Genetic Control Systems Research Articles

A Synthetic Biology Approach to Transgene Expression in Insects.

The ability to control gene expression is pivotal in genetic engineering and synthetic biology. However, in most nonmodel and pest insect species, empirical evidence for predictable modulation of gene expression levels is lacking. This knowledge gap is critical for genetic control systems, particularly in mosquitoes, where transgenic methods offer novel routes for pest control. Commonly, the choice of RNA polymerase II promoter (Pol II) is the primary method for controlling gene expression, but the options are limited. To address this, we developed a systematic approach to characterize modifications in translation initiation sequences (TIS) and 3' untranslated regions (UTR) of transgenes, enabling the creation of a toolbox for gene expression modulation in mosquitoes and potentially other insects. The approach demonstrated highly predictable gene expression changes across various cell lines and 5' regulatory sequences, representing a significant advancement in mosquito synthetic biology gene expression tools.

MGDrivE 3: A decoupled vector-human framework for epidemiological simulation of mosquito genetic control tools and their surveillance.

Novel mosquito genetic control tools, such as CRISPR-based gene drives, hold great promise in reducing the global burden of vector-borne diseases. As these technologies advance through the research and development pipeline, there is a growing need for modeling frameworks incorporating increasing levels of entomological and epidemiological detail in order to address questions regarding logistics and biosafety. Epidemiological predictions are becoming increasingly relevant to the development of target product profiles and the design of field trials and interventions, while entomological surveillance is becoming increasingly important to regulation and biosafety. We present MGDrivE 3 (Mosquito Gene Drive Explorer 3), a new version of a previously-developed framework, MGDrivE 2, that investigates the spatial population dynamics of mosquito genetic control systems and their epidemiological implications. The new framework incorporates three major developments: i) a decoupled sampling algorithm allowing the vector portion of the MGDrivE framework to be paired with a more detailed epidemiological framework, ii) a version of the Imperial College London malaria transmission model, which incorporates age structure, various forms of immunity, and human and vector interventions, and iii) a surveillance module that tracks mosquitoes captured by traps throughout the simulation. Example MGDrivE 3 simulations are presented demonstrating the application of the framework to a CRISPR-based homing gene drive linked to dual disease-refractory genes and their potential to interrupt local malaria transmission. Simulations are also presented demonstrating surveillance of such a system by a network of mosquito traps. MGDrivE 3 is freely available as an open-source R package on CRAN (https://cran.r-project.org/package=MGDrivE2) (version 2.1.0), and extensive examples and vignettes are provided. We intend the software to aid in understanding of human health impacts and biosafety of mosquito genetic control tools, and continue to iterate per feedback from the genetic control community.

A Computational Modeling Approach for the Design of Genetic Control Systems that Respond to Transcriptional Activity.

Recent progress in synthetic biology has enabled the design of complex genetic circuits that interface with innate cellular functions, such as gene transcription, and control user-defined outputs. Implementing these genetic networks in mammalian cells, however, is a cumbersome process that requires several steps of optimization and benefits from the use of predictive modeling. Combining deterministic mathematical models with software-based numerical computing platforms allows researchers to quickly design, evaluate, and optimize multiple circuit topologies to establish experimental constraints that generate the desired control systems. In this chapter, we present a systematic approach based on predictive mathematical modeling to guide the design and construction of gene activity-based sensors. This approach enables user-driven circuit optimization through iterations of sensitivity analyses and parameter scans, providing a universal method to engineer sense and respond cells for diverse applications.

МОДЕЛИРОВАНИЕ СИСТЕМЫ ЭПИГЕНЕТИЧЕСКОЙ ПАМЯТИ С ТРАНСКРИПЦИОННОЙ РЕПРЕССИЕЙ И МЕТИЛИРОВАНИЕМ ДНК

The in silico model of the epigenetic memory system synthesized in Escherichia coli [Maier et al., 2017, Nat. Comm., 8:15336] was developed. The system includes an artificial memory operon with DNA methyltransferase CcrM from Caulobacter crescentus methylating the promoter of this operon, and an engineered transcriptional repressor gene ZnF that blocks the same promoter in an unmethylated state. The memory operon, which is located on the maintenance plasmid, is controlled by a trigger plasmid carrying a specific arabinose-inducible operon. The artificial trigger operon functions as part of an expanded arabinose regulon. An additional model is used to describe the operation of the latter. The unified model of the memory control system takes into account positive and negative control by the regulatory protein araC, negative feedback self-regulation and negative direct, methylation-sensitive regulation by the ZnF protein, and DNA methylation. The models are built in the language of the formalism of the dynamic theory of molecular genetic control systems. The models were calculated in the SETIES software package to analyze the dynamics of control gene networks, dynamic epigenes and cell ensembles. Computer experiments have shown that the switch has two alternative functional modes, each of which is stable in a series of successive cell divisions. Both modes are characterized in terms of the molecular component dynamics of the artificial memory operon, the trigger operon, and the natural L-arabinose catabolism operon.

A programmable protease-based protein secretion platform for therapeutic applications.

Cell-based therapies represent potent enabling technologies in biomedical science. However, current genetic control systems for engineered-cell therapies are predominantly based on the transcription or translation of therapeutic outputs. Here we report a protease-based rapid protein secretion system (PASS) that regulates the secretion of pretranslated proteins retained in the endoplasmic reticulum (ER) owing to an ER-retrieval signal. Upon cleavage by inducible proteases, these proteins are secreted. Three PASS variants (chemPASS, antigenPASS and optoPASS) are developed. With chemPASS, we demonstrate the reversal of hyperglycemia in diabetic mice within minutes via drug-induced insulin secretion. AntigenPASS-equipped cells recognize the tumor antigen and secrete granzyme B and perforin, inducing targeted cell apoptosis. Finally, results from mouse models of diabetes, hypertension and inflammatory pain demonstrate light-induced, optoPASS-mediated therapeutic peptide secretion within minutes, conferring anticipated therapeutic benefits. PASS is a flexible platform for rapid delivery of therapeutic proteins that can facilitate the development and adoption of cell-based precision therapies.

A cybergenetic framework for engineering intein-mediated integral feedback control systems

The ability of biological systems to tightly regulate targeted variables, despite external and internal disturbances, is known as Robust Perfect Adaptation (RPA). Achieved frequently through biomolecular integral feedback controllers at the cellular level, RPA has important implications for biotechnology and its various applications. In this study, we identify inteins as a versatile class of genetic components suitable for implementing these controllers and present a systematic approach for their design. We develop a theoretical foundation for screening intein-based RPA-achieving controllers and a simplified approach for modeling them. We then genetically engineer and test intein-based controllers using commonly used transcription factors in mammalian cells and demonstrate their exceptional adaptation properties over a wide dynamic range. The small size, flexibility, and applicability of inteins across life forms allow us to create a diversity of genetic RPA-achieving integral feedback control systems that can be used in various applications, including metabolic engineering and cell-based therapy.

Viruses, immunity and evolution

In this article, the evolution of viruses is analyzed in terms of their complexity. It is shown that the evolution of viruses is a partially directed process. The participation of viruses and mobile genetic elements in the evolution of other organisms by integration into the genome is also an a priori directed process. The high variability of genomes (including the genes of antibodies), which differs by orders of magnitude for various viruses and their hosts, is not a random process but is the result of the action of a molecular genetic control system. Herein, a model of partially directed evolution of viruses is proposed.Throughout the life cycle of viruses, there is an interaction of complex biologically important molecules that cannot be explained on the basis of classic laws. The interaction of a virus with a cell is essentially a quantum event, including selective long-range action. Such an interaction can be interpreted as the “remote key-lock” principle. In this article, a model of the interaction of biologically important viral molecules with cellular molecules based on nontrivial quantum interactions is proposed. Experiments to test the model are also proposed.

Cybergenetics: Theory and Applications of Genetic Control Systems

There is no design template more important than DNA. Within the sequences of this exquisite substance lie the design plans for each of us and for every living organism. Shaped over billions of years by the creative machinations of evolution, this design template encodes the most complex dynamical systems known to us. Yet, it is only in our lifetimes that we are able to directly edit this template and engineer our own designs. The story that I tell in this article is about our early attempts to design and commission our own control systems in living cells. Guided by what we have learned from controlling man-made systems, we are beginning to develop the theory and methodologies needed to build control systems at the molecular level, an endeavor that is as challenging as it is rewarding. If carried out responsibly, this new ability to reshape the DNA template can have a tremendous benefit for our health and well-being, and will drive major advances in basic science, industrial biotechnology, and medical therapy. In this article, I will take the readers of the proceedings on a journey through the new and promising world of rationally designed genetic control systems. Using a minimum of jargon, I will introduce them to the biological concepts needed to develop an understanding and appreciation of the main design concepts emerging in this nascent area of research. My goal is to convey my own sense of excitement about the possibilities, but it is also to impart a feeling of the opportunities that lay ahead for members of the IEEE to contribute with their own creative ideas to the shaping of this most versatile of design templates, the DNA.

Synthetic Morphogenesis: introducing IEEE journal readers to programming living mammalian cells to make structures.

Synthetic morphogenesis is a new engineering discipline, in which cells are genetically engineered to make designed shapes and structures. At least in this early phase of the field, devices tend to make use of natural shape-generating processes that operate in embryonic development, but invoke them artificially at times and in orders of a technologist's choosing. This requires construction of genetic control, sequencing and feedback systems that have close parallels to electronic design, which is one reason the field may be of interest to readers of IEEE journals. The other reason is that synthetic morphogenesis allows the construction of two-way interfaces, especially opto-genetic and opto-electronic, between the living and the electronic, allowing unprecedented information flow and control between the two types of 'machine'. This review introduces synthetic morphogenesis, illustrates what has been achieved, drawing parallels wherever possible between biology and electronics, and looks forward to likely next steps and challenges to be overcome.

Suppressing mosquito populations with precision guided sterile males

The mosquito Aedes aegypti is the principal vector for arboviruses including dengue/yellow fever, chikungunya, and Zika virus, infecting hundreds of millions of people annually. Unfortunately, traditional control methodologies are insufficient, so innovative control methods are needed. To complement existing measures, here we develop a molecular genetic control system termed precision-guided sterile insect technique (pgSIT) in Aedes aegypti. PgSIT uses a simple CRISPR-based approach to generate flightless females and sterile males that are deployable at any life stage. Supported by mathematical models, we empirically demonstrate that released pgSIT males can compete, suppress, and even eliminate mosquito populations. This platform technology could be used in the field, and adapted to many vectors, for controlling wild populations to curtail disease in a safe, confinable, and reversible manner.

Genetic Programming Guidance Control System for a Reentry Vehicle under Uncertainties

As technology improves, the complexity of controlled systems increases as well. Alongside it, these systems need to face new challenges, which are made available by this technology advancement. To overcome these challenges, the incorporation of AI into control systems is changing its status, from being just an experiment made in academia, towards a necessity. Several methods to perform this integration of AI into control systems have been considered in the past. In this work, an approach involving GP to produce, offline, a control law for a reentry vehicle in the presence of uncertainties on the environment and plant models is studied, implemented and tested. The results show the robustness of the proposed approach, which is capable of producing a control law of a complex nonlinear system in the presence of big uncertainties. This research aims to describe and analyze the effectiveness of a control approach to generate a nonlinear control law for a highly nonlinear system in an automated way. Such an approach would benefit the control practitioners by providing an alternative to classical control approaches, without having to rely on linearization techniques.

HEADING CONTROL OF AUTOMATED GUIDED VEHICLES

With rapid developments in industry wide automation control systems, there have been numerous proposed design and control strategies as alternatives to conventional methods. This term paper deals with the same in the context of Automated Guided Vehicles (AGV’s) with a review of the current literature for proposed design improvements as well as trajectory control systems that can improve productivity and reduce conflicts compared to current methods. The results of these proposed methods are also compared in contrast to each other to obtain holistic view of the merits and demerits of the same. It is found that a fuzzy logic based PID control is a definite upgrade over conventional PID control systems and that the application of LIDAR and Camera based technologies can help enable free roaming AGV’s lending to increased robustness and flexibility. For a control system consisting of a single AGV and multiple processing stations, a genetic algorithm enhanced centralized fuzzy logic control system is the most optimal path planning algorithm whereas in the cases of multiple robots, a dynamic priority allocated free roaming AGVsystem with semi-decentralized control is found to be much more efficient at simultaneously solving paths and reducingconflicts with other robots in real time compared to the purely centralized systems.

Regulation of Phaeodactylum plastid gene transcription by redox, light, and circadian signals.

Diatoms are a diverse group of photosynthetic unicellular algae with a plastid of red-algal origin. As prolific primary producers in the ocean, diatoms fix as much carbon as all rainforests combined. The molecular mechanisms that contribute to the high photosynthetic productivity and ecological success of diatoms are however not yet fully understood. Using the model diatom Phaeodactylum tricornutum, here we show rhythmic transcript accumulation of plastid psaA, psbA, petB, and atpB genes as driven by a free running circadian clock. Treatment with the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea overrides the circadian signal by markedly downregulating transcription of psaA, petB, and atpB genes but not the psbA gene. Changes in light quantity produce little change in plastid gene transcription while the effect of light quality seems modest with only the psaA gene responding in a pattern that is dependent on the redox state of the plastoquinone pool. The significance of these plastid transcriptional responses and the identity of the underlying genetic control systems are discussed with relevance to diatom photosynthetic acclimation.

A versatile genetic control system in mammalian cells and mice responsive to clinically licensed sodium ferulate.

Dynamically adjustable gene- and cell-based therapies are recognized as next-generation medicine. However, the translation of precision therapies into clinics is limited by lack of specific switches controlled by inducers that are safe and ready for clinical use. Ferulic acid (FA) is a phytochemical with a wide range of therapeutic effects, and its salt sodium ferulate (SF) is used as an antithrombotic drug in clinics. Here, we describe an FA/SF-adjustable transcriptional switch controlled by the clinically licensed drug SF. We demonstrated that SF-responsive switches can be engineered to control CRISPR-Cas9 systems for on-command genome/epigenome engineering. In addition, we integrated FA-controlled switches into programmable biocomputers to process logic operations. We further demonstrated the dose-dependent SF-inducible transgene expression in mice by oral administration of SF tablets. Engineered switches responsive to small-molecule clinically licensed drugs to achieve adjustable transgene expression profiles provide new opportunities for dynamic interventions in gene- and cell-based precision medicine.

The Modification of Regulatory Circuits Involved in the Control of Polyhydroxyalkanoates Metabolism to Improve Their Production.

Poly-(3-hydroxyalkanoates) (PHAs) are bacterial carbon and energy storage compounds. These polymers are synthesized under conditions of nutritional imbalance, where a nutrient is growth-limiting while there is still enough carbon source in the medium. On the other side, the accumulated polymer is mobilized under conditions of nutrient accessibility or by limitation of the carbon source. Thus, it is well known that the accumulation of PHAs is affected by the availability of nutritional resources and this knowledge has been used to establish culture conditions favoring high productivities. In addition to this effect of the metabolic status on PHAs accumulation, several genetic regulatory networks have been shown to drive PHAs metabolism, so the expression of the PHAs genes is under the influence of global or specific regulators. These regulators are thought to coordinate PHAs synthesis and mobilization with the rest of bacterial physiology. While the metabolic and biochemical knowledge related to the biosynthesis of these polymers has led to the development of processes in bioreactors for high-level production and also to the establishment of strategies for metabolic engineering for the synthesis of modified biopolymers, the use of knowledge related to the regulatory circuits controlling PHAs metabolism for strain improvement is scarce. A better understanding of the genetic control systems involved could serve as the foundation for new strategies for strain modification in order to increase PHAs production or to adjust the chemical structure of these biopolymers. In this review, the regulatory systems involved in the control of PHAs metabolism are examined, with emphasis on those acting at the level of expression of the enzymes involved and their potential modification for strain improvement, both for higher titers, or manipulation of polymer properties. The case of the PHAs producer Azotobacter vinelandii is taken as an example of the complexity and variety of systems controlling the accumulation of these interesting polymers in response to diverse situations, many of which could be engineered to improve PHAs production.

Towards Universal Control System for Powered Ankle–Foot Prosthesis: A Simulation Study

In powered ankle–foot prostheses, multilevel hierarchical control systems are usually used with predetermined parameters (tuned during the prosthesis trial). Therefore, control systems cannot adaptively interact with terrains variation where control systems is most effective for ground level walking and less effective for ascending or descending stair/slop. In order to address the control system performance in ever-changing terrains, an adaptive mechanism should be included the control system structure. Here, we present a pilot study to illustrate the applicability of a genetic algorithm-based adaptive fuzzy logic control system. The design method could be divided into two stages: initial knowledge base and membership functions for the genetic pool on the basis of the analysis of biological ankle–foot behaviour. Additionally, the construction of genetic optimization mechanism rules and constraints (fitness function, mutation rats, replacement rate, etc.). Takagi–Sugeno–Kang fuzzy (TSK-fuzzy) inference system is selected because the system structure could depict the character of simple impedance control system. The control system and dynamic model were developed using C code and evaluated using MATLAB/Simulink (2019a).

Identification and characterization of the vasa gene in the diamondback moth, Plutella xylostella

Vasa is an ATP-dependent RNA helicase, participating in multiple biological processes. It has been widely used as a germ cell marker and its promoter has become a key component of several genetic pest control systems. Here we present the vasa gene structure and its promoter activity in Plutella xylostella, one of the most destructive pests of cruciferous crops. Full length Pxvasa cDNA sequences were obtained, revealing 14 exons and at least 30 alternatively spliced transcripts. Inferred amino acid sequences showed nine conserved DEAD-box family protein motifs with partial exclusion from some isoforms. Real-time quantitative PCR indicated the up-regulation of Pxvasa in both female and male adults compared with other developmental stages, and the expression levels of Pxvasa were found to be much higher in adult gonads, especially ovaries, than in other tissues. The putative promoter region of Pxvasa was sequenced and several ecdysone-induced transcription factor (TF) binding sites were predicted in silico. To further analyze the promoter region, two upstream regulatory fragments of different lengths were tested as putative promoters in transient cell and embryo expression assays, one of which was subsequently utilized to drive Cas9 expression in vivo. A transgenic line was recovered and the expression patterns of Cas9 and native Pxvasa were profiled in adult tissues and eggs with RT-PCR. This work provides the foundation for further studies on the gene functions of Pxvasa as well as the potential application of its promoter in genetic manipulation of P. xylostella.

A green tea-triggered genetic control system for treating diabetes in mice and monkeys.

Cell-based therapies are recognized as the next frontier in medicine, but the translation of many promising technologies into the clinic is currently limited by a lack of remote-control inducers that are safe and can be tightly regulated. Here, we developed therapeutically active engineered cells regulated by a control system that is responsive to protocatechuic acid (PCA), a metabolite found in green tea. We constructed multiple genetic control technologies that could toggle a PCA-responsive ON/OFF switch based on a transcriptional repressor from Streptomyces coelicolor We demonstrated that PCA-controlled switches can be used for guide RNA expression-mediated control of the CRISPR-Cas9 systems for gene editing and epigenetic remodeling. We showed how these technologies could be used as implantable biocomputers in live mice to perform complex logic computations that integrated signals from multiple food metabolites. Last, we used our system to treat type 1 and type 2 diabetes in mice and cynomolgus monkeys. This biocompatible and versatile food phenolic acid-controlled transgenic device opens opportunities for dynamic interventions in gene- and cell-based precision medicine.

A methodical approach for evaluating the variability of productivity and fruit quality in the genetic collections of strawberry (Fragaria × ananassa Duch.)

For strawberry (Fragaria × ananassa Duch., 2n = 8x = 56), which is the leading berry crop in the world, research into the genotype × environment interaction is important. A complicated genomic composition, the diversity of genetic control systems, and a strong modifying effect of growing conditions on the implementation of quantitative traits make it necessary to improve methods for analysis of the genotypic variability of economically valuable traits with the aim of identifying genotypes that are characterized by stability and adaptive qualities in a wide ecological range of growing conditions. In 2016–2018, twenty-seven strawberry varieties were studied in the collections of North Caucasian Federal Scientific Center of Horticulture, Viticulture and Krymsk Experiment Breeding Station, VIR Branch. Field experiments and data counts were set and carried out according to a single scheme. The following characteristics were studied: the number of inflorescences (units per plant), the number of berries (units per plant), the average weight of berry and berry of the first order (g), total and marketable yield (g per plant), firmness of fruit (g), sugar content in berries on Degrees Brix (°Bx), sugar-acid index. The purpose of this work was the development of a methodical approach to assessing the contribution of the genotype– environment interaction to the variability of the traits of productivity and fruit quality and the determination of strawberry varieties with a stable genotype. To this end, the mathematical models of two- and three-factor analysis of variance and cluster analysis using Ward’s method were employed. According to the results of this work, strawberry varieties grown in different climatic conditions show differences in the structure of the variability of the traits of productivity and fruit quality. For the conditions of the city of Krymsk, the influence of the genotype of the variety was predominant, and for the conditions of the city of Krasnodar, in addition to the influence of the genotype of the variety, the environmental component in the form of the genotype–environment interaction is also significant. A statistically significant influence of the growing zone has been established for the traits of productivity and fruit quality, with the exception of the average weight of fruit. At the same time, differences in the mean values of the traits of varieties can be both significant and partially or completely absent. To identify varieties with promise for cultivation in the areas studied, it is recommended to use cluster analysis on the informative complex of traits with the calculation of the Euclidean distances for varieties that were grown under different conditions. The magnitude of the Euclidean distance will be the measure of the influence of a particular environment on the genotype of plants. The smaller the value of the Euclidean distance in a variety, according to the complex of the traits studied, the more stable this variety is.

Fulfilling the responsibilites of the manager of the laboratory for the selection of forage crops

The effects of GCA samples according to the results of dialysis analysis are given. Genetic control systems of traits of each variety are reflected, in particular the relative contribution of dominant and recessive alleles. According to the analysis of the effects of general combinatorial ability, samples with high indicators were selected for most of the studied features: the number of vegetatively elongated shoots - Poltava 52; foliage - Anto; dry matter yield - Poltava 52; protein content in dry matter - Poltava 52; the number of generative shoots - Anto and Poltava 52; panicle length - Anto and Radio-mutant k-7; seed productivity - Anto. A high level of the coefficients of heredity of the studied signs in a broad sense (H2 = 0,93 – 0,99) have been established. The coefficients of heredity in the narrow sense (h2) in terms of genetic variability due to additive effects of genes were different. The highest rate was in seed productivity (0,91), the number of vegetatively elongated shoots (0,78), dry matter yield (0,70); the lowest - in the length of the panicle (0,35) and the height of the plant - (0,43). The protein content was average (0,58). Key words: unbearded hundred-spike, diallel crosses, general combination ability, heredity.