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Synthetic biology aims to engineer biological systems for customized tasks through the bottom-up assembly of fundamental building blocks, which requires high-quality libraries of reliable, modular, and standardized genetic parts. To establish sets of parts that work well together, synthetic biologists created standardized part libraries in which every component is analyzed in the same metrics and context. Here we present a state-of-the-art review of the currently available part libraries for designing biocircuits and their gene expression regulation paradigms at transcriptional, translational, and post-translational levels in Escherichia coli . We discuss the necessary facets to integrate these parts into complex devices and systems along with the current efforts to catalogue and standardize measurement data. To better display the range of available parts and to facilitate part selection in synthetic biology workflows, we established biopartsDB, a curated database of well-characterized and useful genetic part and device libraries with detailed quantitative data validated by the published literature.

As synthetic biology permeates society, the signal processing circuits in engineered living systems must be customized to meet practical demands. Towards this mission, novel regulatory mechanisms and genetic circuits with unprecedented complexity have been implemented over the past decade. These regulatory mechanisms, such as transcription and translation control, could be integrated into hybrid circuits termed "multi-level circuits". The multi-level circuit design will tremendously benefit the current genetic circuit design paradigm, from modifying basic circuit dynamics to facilitating real-world applications, unleashing our capabilities to customize cellular signal processing and address global challenges through synthetic biology., (© 2023. The Author(s).)

Introduction: Bacterial synthetic biology has provided powerful tools to revolutionize the drug discovery process. These tools can be harnessed to generate bacterial novel pharmaceutical compounds with enhanced bioactivity and selectivity or to create genetically modified microorganisms as living drugs., Areas Covered: This review provides a current overview of the state-of-the-art in bacterial synthetic biology tools for novel drug discovery. The authors discuss the application of these tools including bioinformatic tools, CRISPR tools, engineered bacterial transcriptional regulators, and synthetic biosensors for novel drug discovery. Additionally, the authors present the recent progress on reprogramming bacteriophages as living drugs to fight against antibiotic-resistant pathogens., Expert Opinion: The field of using bacterial synthetic biology tools for drug discovery is rapidly advancing. However, challenges remain in developing reliable and robust methods to engineer bacteria. Further advancements in synthetic biology hold promise to speed up drug discovery, facilitating the development of novel therapeutics against various diseases.

Synthetic biology offers new tools and capabilities of engineering cells with desired functions for example as new biosensing platforms leveraging engineered microbes. In the last two decades, bacterial cells have been programmed to sense and respond to various input cues for versatile purposes including environmental monitoring, disease diagnosis and adaptive biomanufacturing. Despite demonstrated proof-of-concept success in the laboratory, the real-world applications of microbial sensors have been restricted due to certain technical and societal limitations. Yet, most limitations can be addressed by new technological developments in synthetic biology such as circuit design, biocontainment and machine learning. Here, we summarize the latest advances in synthetic biology and discuss how they could accelerate the development, enhance the performance and address the present limitations of microbial sensors to facilitate their use in the field. We view that programmable living sensors are promising sensing platforms to achieve sustainable, affordable and easy-to-use on-site detection in diverse settings., (© 2021 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Nitric oxide (NO) is an important disease biomarker found in many chronic inflammatory diseases and cancers. A well-characterized nitric sensing system is useful to aid the rapid development of bacteria therapy and synthetic biology. In this work, we engineered a set of NO-responsive biosensors based on the P norV promoter and its NorR regulator in the norRVW operon; the circuits were characterized and optimized in probiotic Escherichia coli Nissle 1917 and mini SimCells (minicells containing designed gene circuits for specific tasks). Interestingly, the expression level of NorR displayed an inverse correlation to the P norV promoter activation, as a strong expression of the NorR regulator resulted in a low amplitude of NO-inducible gene expression. This could be explained by a competitive binding mechanism where the activated and inactivated NorR competitively bind to the same site on the P norV promoter. To overcome such issues, the NO induction performance was further improved by making a positive feedback loop that fine-tuned the level of NorR. In addition, by examining two integration host factor (IHF) binding sites of the P norV promoter, we demonstrated that the deletion of the second IHF site increased the maximum signal output by 25% (500 μM DETA/NO) with no notable increase in the basal expression level. The optimized NO-sensing gene circuit in anucleate mini SimCells exhibited increased robustness against external fluctuation in medium composition. The NO detection limit of the optimized gene circuit pPnorVβ was also improved from 25.6 to 1.3 nM in mini SimCells. Moreover, lyophilized mini SimCells can maintain function for over 2 months. Hence, SimCell-based NO biosensors could be used as safe sensor chassis for synthetic biology.

The front cover artwork is provided by the group of Dr. Baojun Wang (The University of Edinburgh). The image shows an engineered bacterial cell containing a genetic amplifier circuit which transforms a weak input signal into a larger easily detectable output signal. The electronics symbols used to illustrate the genetic circuit highlight the programmability of the circuit components enabled by state-of-the-art synthetic biology tools. Read the full text of the Review at 10.1002/cphc.201900739., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recent advances in synthetic biology research have been underpinned by an exponential increase in available genomic information and a proliferation of advanced DNA assembly tools. The adoption of plasmid vector assembly standards and parts libraries has greatly enhanced the reproducibility of research and the exchange of parts between different labs and biological systems. However, a standardized modular cloning (MoClo) system is not yet available for cyanobacteria, which lag behind other prokaryotes in synthetic biology despite their huge potential regarding biotechnological applications. By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile system called CyanoGate that unites cyanobacteria with plant and algal systems. Here, we describe the generation of a suite of parts and acceptor vectors for making (1) marked/unmarked knock-outs or integrations using an integrative acceptor vector, and (2) transient multigene expression and repression systems using known and previously undescribed replicative vectors. We tested and compared the CyanoGate system in the established model cyanobacterium Synechocystis sp. PCC 6803 and the more recently described fast-growing strain Synechococcus elongatus UTEX 2973. The UTEX 2973 fast-growth phenotype was only evident under specific growth conditions; however, UTEX 2973 accumulated high levels of proteins with strong native or synthetic promoters. The system is publicly available and can be readily expanded to accommodate other standardized MoClo parts to accelerate the development of reliable synthetic biology tools for the cyanobacterial community., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

A central aim of synthetic biology is to build organisms that can perform useful activities in response to specified conditions. The digital computing paradigm which has proved so successful in electrical engineering is being mapped to synthetic biological systems to allow them to make such decisions. However, stochastic molecular processes have graded input-output functions, thus, bioengineers must select those with desirable characteristics and refine their transfer functions to build logic gates with digital-like switching behaviour. Recent efforts in genome mining and the development of programmable RNA-based switches, especially CRISPRi, have greatly increased the number of parts available to synthetic biologists. Improvements to the digital characteristics of these parts are required to enable robust predictable design of deeply layered logic circuits., (Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Synthetic biologists aim to construct novel genetic circuits with useful applications through rational design and forward engineering. Given the complexity of signal processing that occurs in natural biological systems, engineered microbes have the potential to perform a wide range of desirable tasks that require sophisticated computation and control. Realising this goal will require accurate predictive design of complex synthetic gene circuits and accompanying large sets of quality modular and orthogonal genetic parts. Here we present a current overview of the versatile components and tools available for engineering gene circuits in microbes, including recently developed RNA-based tools that possess large dynamic ranges and can be easily programmed. We introduce design principles that enable robust and scalable circuit performance such as insulating a gene circuit against unwanted interactions with its context, and we describe efficient strategies for rapidly identifying and correcting causes of failure and fine-tuning circuit characteristics., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Microorganisms are able to respond effectively to diverse signals from their environment and internal metabolism owing to their inherent sophisticated information processing capacity. A central aim of synthetic biology is to control and reprogramme the signal processing pathways within living cells so as to realise repurposed, beneficial applications ranging from disease diagnosis and environmental sensing to chemical bioproduction. To date most examples of synthetic biological signal processing have been built based on digital information flow, though analogue computing is being developed to cope with more complex operations and larger sets of variables. Great progress has been made in expanding the categories of characterised biological components that can be used for cellular signal manipulation, thereby allowing synthetic biologists to more rationally programme increasingly complex behaviours into living cells. Here we present a current overview of the components and strategies that exist for designer cell signal processing and decision making, discuss how these have been implemented in prototype systems for therapeutic, environmental, and industrial biotechnological applications, and examine emerging challenges in this promising field., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Biosensors are analytical devices that utilize biological sensing elements, such as enzymes, antibodies, nucleic acids, or cells, to detect a given analyte [...].

Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. To address this, we augment a mini-Mu transposon-based screening approach and devise the intein-assisted bisection mapping (IBM) method. IBM robustly reveals clusters of split sites on five proteins, converting them into AND or NAND logic gates. We further show that the use of inteins expands functional sequence space for splitting a protein. We also demonstrate the utility of our approach over rational inference of split sites from secondary structure alignment of homologous proteins, and that basal activities of highly active proteins can be mitigated by splitting them. Our work offers a generalizable and systematic route towards creating split protein-intein fusions for synthetic biology. Split inteins are powerful tools for designing synthetic split proteins. Here the authors use a mini-Mu transposon screen to map split sites, enabling the development of protein-based logic gates and fine control of protein activity. [ABSTRACT FROM AUTHOR]

Cyanobacteria utilize sunlight to convert carbon dioxide into a wide variety of secondary metabolites and show great potential for green biotechnology applications. Although cyanobacterial synthetic biology is less mature than for other heterotrophic model organisms, there are now a range of molecular tools available to modulate and control gene expression. One area of gene regulation that still lags behind other model organisms is the modulation of gene transcription, particularly transcription termination. A vast number of intrinsic transcription terminators are now available in heterotrophs, but only a small number have been investigated in cyanobacteria. As artificial gene expression systems become larger and more complex, with short stretches of DNA harboring strong promoters and multiple gene expression cassettes, the need to stop transcription efficiently and insulate downstream regions from unwanted interference is becoming more important. In this study, we adapted a dual reporter tool for use with the CyanoGate MoClo Assembly system that can quantify and compare the efficiency of terminator sequences within and between different species. We characterized 34 intrinsic terminators in Escherichia coli , Synechocystis sp. PCC 6803, and Synechococcus elongatus UTEX 2973 and observed significant differences in termination efficiencies. However, we also identified five terminators with termination efficiencies of >96% in all three species, indicating that some terminators can behave consistently in both heterotrophic species and cyanobacteria. [ABSTRACT FROM AUTHOR]

CRISPR (clustered regularly interspaced short palindromic repeats) activation (CRISPRa) in bacteria is an attractive method for programmable gene activation. Recently, a eukaryote‐like, σ54‐dependent CRISPRa system has been reported. It exhibits high dynamic ranges and permits flexible target site selection. Here, an overview of the existing strategies of CRISPRa in bacteria is presented, and the characteristics and design principles of the CRISPRa system are introduced. Possible scenarios for applying the eukaryote‐like CRISPRa system is discussed with corresponding suggestions for performance optimization and future functional expansion. The authors envision the new eukaryote‐like CRISPRa system enabling novel designs in multiplexed gene regulation and promoting research in the σ54‐dependent gene regulatory networks among a variety of biotechnology relevant or disease‐associated bacterial species. [ABSTRACT FROM AUTHOR]

Inteins are protein segments capable of joining adjacent residues via a peptide bond. In this process known as protein splicing, the intein itself is not present in the final sequence, thus achieving scarless peptide ligation. Here, we assess the splicing activity of 34 inteins (both uncharacterized and known) using a rapid split fluorescent reporter characterization platform, and establish a library of 15 mutually orthogonal split inteins for in vivo applications, 10 of which can be simultaneously used in vitro. We show that orthogonal split inteins can be coupled to multiple split transcription factors to implement complex logic circuits in living organisms, and that they can also be used for the in vitro seamless assembly of large repetitive proteins with biotechnological relevance. Our work demonstrates the versatility and vast potential of an expanded library of orthogonal split inteins for their use in the fields of synthetic biology and protein engineering. Inteins allow the joining of protein segments through scarless ligation. Here the authors assess 34 inteins to establish a library of 15 mutually orthogonal split inteins for in vivo applications, 10 of which are also active in vitro. [ABSTRACT FROM AUTHOR]

Abstract: Cells perceive a wide variety of cellular and environmental signals, which are often processed combinatorially to generate particular phenotypic responses. Here, we employ both single and mixed cell type populations, pre-programmed with engineered modular cell signalling and sensing circuits, as processing units to detect and integrate multiple environmental signals. Based on an engineered modular genetic AND logic gate, we report the construction of a set of scalable synthetic microbe-based biosensors comprising exchangeable sensory, signal processing and actuation modules. These cellular biosensors were engineered using distinct signalling sensory modules to precisely identify various chemical signals, and combinations thereof, with a quantitative fluorescent output. The genetic logic gate used can function as a biological filter and an amplifier to enhance the sensing selectivity and sensitivity of cell-based biosensors. In particular, an Escherichia coli consortium-based biosensor has been constructed that can detect and integrate three environmental signals (arsenic, mercury and copper ion levels) via either its native two-component signal transduction pathways or synthetic signalling sensors derived from other bacteria in combination with a cell-cell communication module. We demonstrate how a modular cell-based biosensor can be engineered predictably using exchangeable synthetic gene circuit modules to sense and integrate multiple-input signals. This study illustrates some of the key practical design principles required for the future application of these biosensors in broad environmental and healthcare areas. [Copyright &y& Elsevier]

Cells live in an ever-changing environment and continuously sense, process and react to environmental signals using their inherent signaling and gene regulatory networks. Recently, there have been great advances on rewiring the native cell signaling and gene networks to program cells to sense multiple noncognate signals and integrate them in a logical manner before initiating a desired response. Here, we summarize the current state-of-the-art of engineering synthetic genetic logic circuits to customize cellular signaling behaviors, and discuss their promising applications in biocomputing, environmental, biotechnological and biomedical areas as well as the remaining challenges in this growing field. [ABSTRACT FROM AUTHOR]

Recent advances in synthetic biology and an emerging algal biotechnology market have spurred a prolific increase in the availability of molecular tools for cyanobacterial research. Nevertheless, work to date has focused primarily on only a small subset of model species, which arguably limits fundamental discovery and applied research towards wider commercialisation. Here, we review the requirements for uptake of new strains, including several recently characterised fast-growing species and promising non-model species. Furthermore, we discuss the potential applications of new techniques available for transformation, genetic engineering and regulation, including an up-to-date appraisal of current Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein (CRISPR/Cas) and CRISPR interference (CRISPRi) research in cyanobacteria. We also provide an overview of several exciting molecular tools that could be ported to cyanobacteria for more advanced metabolic engineering approaches (e.g., genetic circuit design). Lastly, we introduce a forthcoming mutant library for the model species Synechocystis sp. PCC 6803 that promises to provide a further powerful resource for the cyanobacterial research community. [ABSTRACT FROM AUTHOR]

Transcriptional regulation by nuclease-deficient CRISPR/Cas is a popular and valuable tool for routine control of gene expression. CRISPR interference in bacteria can be reliably achieved with high efficiencies. Yet, options for CRISPR activation (CRISPRa) remained limited in flexibility and activity because they relied on σ70 promoters. Here we report a eukaryote-like bacterial CRISPRa system based on σ54-dependent promoters, which supports long distance, and hence multi-input regulation with high dynamic ranges. Our CRISPRa device can activate σ54-dependent promoters with biotechnology relevance in non-model bacteria. It also supports orthogonal gene regulation on multiple levels. Combining our CRISPRa with dxCas9 further expands flexibility in DNA targeting, and boosts dynamic ranges into regimes that enable construction of cascaded CRISPRa circuits. Application-wise, we construct a reusable scanning platform for readily optimizing metabolic pathways without library reconstructions. This eukaryote-like CRISPRa system is therefore a powerful and versatile synthetic biology tool for diverse research and industrial applications. CRISPR activation strategies in bacteria are limited due to the reliance on σ70 promoters. Here the authors demonstrate eukaryote-like gene activation with high dynamic ranges using σ54- dependent promoters. [ABSTRACT FROM AUTHOR]

Wastewater surveillance is a powerful tool to understand community profiling in terms of health monitoring. Tracking biomarkers such as inorganic and organic pollutants, drugs, and pathogens in wastewater gives a general idea about the lifestyle and health status of a population as well as pollutant exposure caused by various toxic chemicals. Notably, tracing pathogenic clues could help predict and prevent disease outbreaks such as the ongoing COVID-19 pandemic in communities. To this end, developing portable biosensing platforms will facilitate the on-site monitoring of water contamination without requiring complex equipment. New technological developments in synthetic biology have advanced both synthetic gene circuit-based biosensors and new in vitro detection strategies coupled with easy-to-interpret visualization methods. Here, we summarize the latest advances in synthetic biology tools and discuss how they enable the development of rapid, low-cost, ease-to-use and field-deployable biosensors for monitoring a variety of water contaminants and health-related biomarkers in the environment. [Display omitted] • Regular rapid wastewater assessment permits early warning of contamination and prevention of disease outbreaks. • Synthetic biology enables the development of field-deployable biosensors for various pathogens and toxins in water. • Synthetic cell-based and cell-free sensing systems are promising tools for wastewater-based epidemiology surveillance. [ABSTRACT FROM AUTHOR]

Routine monitoring of sanitation and hygiene to identify fecal contamination in aquatic environments is an effective means to prevent threatening disease transmission. Compared to immunological or genetic methods performed in a central lab, enzymatic assays are considered simple, quick, cost-effective and thus promising for in-field (near) real-time detection. However, the long detection time for mildly polluted samples is a major obstacle to its deployment as an early warning system. Here, the challenges faced by the assays in real environmental sample measurements are summarized, followed by the current status of their field applications. Furthermore, the likelihood and ways are discussed for significant assay improvements using state-of-the-art synthetic biology technologies. Rapid advances in synthetic biology such as various new enabling tools for precise biomolecular manipulation and cell-free expression systems have great potential to address the present bottlenecks of the enzymatic assays, paving the way for better early warning strategies and performance. • Enzymatic assays for routinely identifying fecal contamination permit efficient prevention of infectious disease outbreaks. • Rapid enzymatic assays face complex challenges in on-site applications for the purpose of early warning. • Synthetic biology enabling tools have potential to significantly improve the sensitivity and rapidity of the enzymatic assays. [ABSTRACT FROM AUTHOR]