a-100-kw-class-applied-field-magnetoplasmadynamic-thruster

A 100 KW Class Applied-field Magnetoplasmadynamic Thruster

Applied-field magnetoplasmadynamic thrusters (AF-MPD thrusters) are hybrid accelerators in which electromagnetic and gas dynamic processes accelerate ...

genetic-modification-cyanobacteria-conjugation-using-cyanogate

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful ...

Bacterial cells continuously sense and respond to their environment using their inherent signalling and gene regulatory networks. Cells are equipped with parallel signalling pathways, which can specifically cope with individual input signals, while interconnectivities between pathways lead to an enhanced complexity of regulatory responses that enable sophisticated adaptation. In principle, any cell signalling pathway may be rewired to respond to non-cognate signals by exchanging and recombining their underlying cognate signalling components. In the present article, we review the engineering strategies and use of chimaeric regulatory proteins in cell signalling pathways, especially the TCS (two-component signalling) system in bacteria, to achieve novel customized signalling or regulatory functions. We envisage that engineered chimaeric regulatory proteins can play an important role to aid both forward and reverse engineering of biological systems for many desired applications. 

Rewiring cell signalling through chimaeric regulatory protein engineering.

The σ(54)-dependent transcription in bacteria requires specific activator proteins, bacterial enhancer binding protein (bEBP), members of the AAA+ (ATPases Associated with various cellular Activities) protein family. The bEBPs usually form oligomers in order to hydrolyze ATP and make open promoter complexes. The bEBP formed by HrpR and HrpS activates transcription from the σ(54)-dependent hrpL promoter responsible for triggering the Type Three Secretion System in Pseudomonas syringae pathovars. Unlike other bEBPs that usually act as homohexamers, HrpR and HrpS operate as a highly co-dependent heterohexameric complex. To understand the organization of the HrpRS complex and the HrpR and HrpS strict co-dependence, we have analyzed the interface between subunits using the random and directed mutagenesis and available crystal structures of several closely related bEBPs. We identified key residues required for the self-association of HrpR (D32, E202 and K235) with HrpS (D32, E200 and K233), showed that the HrpR D32 and HrpS K233 residues form interacting pairs directly involved in an HrpR-HrpS association and that the change in side-chain length at position 233 in HrpS affects self-association and interaction with the HrpR and demonstrated that the HrpS D32, E200 and K233 are not involved in negative regulation imposed by HrpV. We established that the equivalent residues K30, E200 and E234 in a homo-oligomeric bEBP, PspF, are required for the subunit communication and formation of an oligomeric lock that cooperates with the ATP γ-phosphate sensing PspF residue R227, providing insights into their roles in the heteromeric HrpRS co-complex. 

Determination of the self-association residues within a homomeric and a heteromeric AAA+ enhancer binding protein.

Synthetic biology aims to control and reprogram signal processing pathways within living cells so as to realize repurposed, beneficial applications. Here we report the design and construction of a set of modular and gain-tunable genetic amplifiers in Escherichia coli capable of amplifying a transcriptional signal with wide tunable-gain control in cascaded gene networks. The devices are engineered using orthogonal genetic components (hrpRS, hrpV and PhrpL) from the hrp (hypersensitive response and pathogenicity) gene regulatory network in Pseudomonas syringae. The amplifiers can linearly scale up to 21-fold the transcriptional input with a large output dynamic range, yet not introducing significant time delay or significant noise during signal amplification. The set of genetic amplifiers achieves different gains and input dynamic ranges by varying the expression levels of the underlying ligand-free activator proteins in the device. As their electronic counterparts, these engineered transcriptional amplifiers can act as fundamental building blocks in the design of biological systems by predictably and dynamically modulating transcriptional signal flows to implement advanced intra- and extra-cellular control functions. 

Engineering modular and tunable genetic amplifiers for scaling transcriptional signals in cascaded gene networks.

We designed and constructed versatile modular genetic logic gates in bacterial cells. These function as digital logic 1-input Buffer gate, 2-input and 3-input AND gates with one inverted input and integrate multiple chemical input signals in customised logic manners. Such rapidly engineered devices serve to achieve increased sensing signal selectivity. 

Rapid engineering of versatile molecular logic gates using heterologous genetic transcriptional modules.

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. 

Designer cell signal processing circuits for biotechnology.

Ligand-responsive transcription factors in prokaryotes found simple small molecule-inducible gene expression systems. These have been extensively used for regulated protein production and associated biosynthesis of fine chemicals. However, the promoter and protein engineering approaches traditionally used often pose significant restrictions to predictably and rapidly tune the expression profiles of inducible expression systems. Here, we present a new unified and rational tuning method to amplify the sensitivity and dynamic ranges of versatile small molecule-inducible expression systems. We employ a systematic variation of the concentration of intracellular receptors for transcriptional control. We show that a low density of the repressor receptor (e.g. TetR and ArsR) in the cell can significantly increase the sensitivity and dynamic range, whereas a high activator receptor (e.g. LuxR) density achieves the same outcome. The intracellular concentration of receptors can be tuned in both discrete and continuous modes by adjusting the strength of their cognate driving promoters. We exemplified this approach in several synthetic receptor-mediated sensing circuits, including a tunable cell-based arsenic sensor. The approach offers a new paradigm to predictably tune and amplify ligand-responsive gene expression with potential applications in synthetic biology and industrial biotechnology.

Amplification of small molecule-inducible gene  expression via tuning of intracellular receptor  densities.

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. 

Tools and Principles for Microbial Gene Circuit Engineering.

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.

Recognizing and engineering digital-like logic gates and switches in gene regulatory networks.

Bacteria live in an ever changing environment and, to adapt their physiology, they have to sense the changes. Our current understanding of the mechanisms and elements involved in the detection and processing of these environmental signals grant us access to an array of genetic components able to process such information. As engineers can use different electronic components to build a circuit, we can rewire the cellular components to create digital logic and analogue gene circuits that will program cell behaviour in a designed manner in response to a specific stimulus. Here we present the methods and protocols for designing and implementing synthetic cell-based biosensors that use engineered genetic logic and analogue amplifying circuits to significantly increase selectivity and sensitivity, for example, for heavy metal ions in an aqueous environment. The approach is modular and can be readily applied to improving the sensing limit and performance of a range of microbial cell-based sensors to meet their real world detection requirement.

Synthetic Cell-Based Sensors with Programmed Selectivity and Sensitivity.

Synthetic biology approaches commonly introduce heterologous gene networks into a host to predictably program cells, with the expectation of the synthetic network being orthogonal to the host background. However, introduced circuits may interfere with the host's physiology, either indirectly by posing a metabolic burden and/or through unintended direct interactions between parts of the circuit with those of the host, affecting functionality. Here we used RNA-Seq transcriptome analysis to quantify the interactions between a representative heterologous AND gate circuit and the host Escherichia coli under various conditions including circuit designs and plasmid copy numbers. We show that the circuit plasmid copy number outweighs circuit composition for their effect on host gene expression with medium-copy number plasmid showing more prominent interference than its low-copy number counterpart. In contrast, the circuits have a stronger influence on the host growth with a metabolic load increasing with the copy number of the circuits. Notably, we show that variation of copy number, an increase from low to medium copy, caused different types of change observed in the behavior of components in the AND gate circuit leading to the unbalance of the two gate-inputs and thus counterintuitive output attenuation. The study demonstrates the circuit plasmid copy number is a key factor that can dramatically affect the orthogonality, burden and functionality of the heterologous circuits in the host chassis. The results provide important guidance for future efforts to design orthogonal and robust gene circuits with minimal unwanted interaction and burden to their host.

Orthogonality and Burdens of Heterologous AND Gate Gene Circuits in E. coli.

The dimerization and high-order oligomerization of transcription factors has endowed them with cooperative regulatory capabilities that play important roles in many cellular functions. However, such advanced regulatory capabilities have not been fully exploited in synthetic biology and genetic engineering. Here, we engineered a C-terminally fused oligomerization domain to improve the cooperativity of transcription factors. First, we found that two of three designed oligomerization domains significantly increased the cooperativity and ultrasensitivity of a transcription factor for the regulated promoter. Then, seven additional transcription factors were used to assess the modularity of the oligomerization domains, and their ultrasensitivity was generally improved, as assessed by their Hill coefficients. Moreover, we also demonstrated that the allosteric capability of the ligand-responsive domain remained intact when fusing with the designed oligomerization domain. As an example application, we showed that the engineered ultrasensitive transcription factor could be used to significantly improve the performance of a "stripe-forming" gene circuit. We envision that the oligomerization modules engineered in this study could act as a powerful tool to rapidly tune the underlying response profiles of synthetic gene circuits and metabolic pathway controllers.

Engineering the Ultrasensitive Transcription Factors by Fusing a Modular Oligomerization Domain.

Synthetic biology aims to engineer and redesign biological systems for useful real-world applications in biomanufacturing, biosensing and biotherapy following a typical design-build-test cycle. Inspired from computer science and electronics, synthetic gene circuits have been designed to exhibit control over the flow of information in biological systems. Two types are Boolean logic inspired TRUE or FALSE digital logic and graded analog computation. Key principles for gene circuit engineering include modularity, orthogonality, predictability and reliability. Initial circuits in the field were small and hampered by a lack of modular and orthogonal components, however in recent years the library of available parts has increased vastly. New tools for high throughput DNA assembly and characterization have been developed enabling rapid prototyping, systematic in situ characterization, as well as automated design and assembly of circuits. Recently implemented computing paradigms in circuit memory and distributed computing using cell consortia will also be discussed. Finally, we will examine existing challenges in building predictable large-scale circuits including modularity, context dependency and metabolic burden as well as tools and methods used to resolve them. These new trends and techniques have the potential to accelerate design of larger gene circuits and result in an increase in our basic understanding of circuit and host behaviour.

Scaling up genetic circuit design for cellular computing: advances and prospects.

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  sp. PCC 6803 and the more recently described fast-growing strain  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.

CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax.

Cell-based biosensors have great potential to detect various toxic and pathogenic contaminants in aqueous environments. However, frequently they cannot meet practical requirements due to insufficient sensing performance. To address this issue, we investigated a modular, cascaded signal amplifying methodology. We first tuned intracellular sensory receptor densities to increase sensitivity, and then engineered multi-layered transcriptional amplifiers to sequentially boost output expression level. We demonstrated these strategies by engineering ultrasensitive bacterial sensors for arsenic and mercury, and improved detection limit and output up to 5,000-fold and 750-fold, respectively. Coupled by leakage regulation approaches, we developed an encapsulated microbial sensor cell array for low-cost, portable and precise field monitoring, where the analyte can be readily quantified via displaying an easy-to-interpret volume bar-like pattern. The ultrasensitive signal amplifying methodology along with the background regulation and the sensing platform will be widely applicable to many other cell-based sensors, paving the way for their real-world applications.

Cascaded amplifying circuits enable ultrasensitive cellular sensors for toxic metals.

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 σ promoters. Here we report a eukaryote-like bacterial CRISPRa system based on σ-dependent promoters, which supports long distance, and hence multi-input regulation with high dynamic ranges. Our CRISPRa device can activate σ-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.

Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria.

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  sp. PCC 6803 that promises to provide a further powerful resource for the cyanobacterial research community.

Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology.

Cell-based biosensors offer cheap, portable and simple methods of detecting molecules of interest but have yet to be truly adopted commercially. Issues with their performance and specificity initially slowed the development of cell-based biosensors. With the development of rational approaches to tune response curves, the performance of biosensors has rapidly improved and there are now many biosensors capable of sensing with the required performance. This has stimulated an increased interest in biosensors and their commercial potential. However the reliability, long term stability and biosecurity of these sensors are still barriers to commercial application and public acceptance. Research into overcoming these issues remains active. Here we present the state-of-the-art tools offered by synthetic biology to allow construction of cell-based biosensors with customisable performance to meet the real world requirements in terms of sensitivity and dynamic range and discuss the research progress to overcome the challenges in terms of the sensor stability and biosecurity fears.

ArticleTotal : 2	Year
Publication title Cited by 9	2018
Publication title Cited by 19	2019

Article	Year
Rewiring cell signalling through chimaeric regulatory protein engineering. Biochemical Society transactions\| PubMed ID: 24059508	2013
Determination of the self-association residues within a homomeric and a heteromeric AAA+ enhancer binding protein. Journal of molecular biology\| PubMed ID: 24434682	2014
Engineering modular and tunable genetic amplifiers for scaling transcriptional signals in cascaded gene networks. Nucleic acids research\| PubMed ID: 25030903	2014
Rapid engineering of versatile molecular logic gates using heterologous genetic transcriptional modules. Chemical communications (Cambridge, England)\| PubMed ID: 25062273	2014
Designer cell signal processing circuits for biotechnology. New biotechnology\| PubMed ID: 25579192	2015
Amplification of small molecule-inducible gene expression via tuning of intracellular receptor densities. Nucleic acids research\| PubMed ID: 25589545	2015
Tools and Principles for Microbial Gene Circuit Engineering. Journal of molecular biology\| PubMed ID: 26463592	2016
Recognizing and engineering digital-like logic gates and switches in gene regulatory networks. Current opinion in microbiology\| PubMed ID: 27450541	2016
Synthetic Cell-Based Sensors with Programmed Selectivity and Sensitivity. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 28299699	2017
Orthogonality and Burdens of Heterologous AND Gate Gene Circuits in E. coli. ACS synthetic biology\| PubMed ID: 29240998	2018
Engineering the Ultrasensitive Transcription Factors by Fusing a Modular Oligomerization Domain. ACS synthetic biology\| PubMed ID: 29733626	2018
Scaling up genetic circuit design for cellular computing: advances and prospects. Natural computing\| PubMed ID: 30524216	2018
CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax. Plant physiology\| PubMed ID: 30819783	2019
Cascaded amplifying circuits enable ultrasensitive cellular sensors for toxic metals. Nature chemical biology\| PubMed ID: 30911179	2019
Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria. Nature communications\| PubMed ID: 31451697	2019
Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology. Microorganisms\| PubMed ID: 31569579	2019
Synthetic Biology Enables Programmable Cell-Based Biosensors. Chemphyschem : a European journal of chemical physics and physical chemistry\| PubMed ID: 31585026	2019

Baojun Wang

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