In vivo biosensors: mechanisms,development, and applications

In vivo biosensors can recognize and respond to specific cellular stimuli. In recent years, biosensors have been increasingly used in metabolic engineering and synthetic biology, because they can be implemented in synthetic circuits to control the expression of reporter genes in response to specific cellular stimuli, such as a certain metabolite or a change in pH. There are many types of natural sensing devices, which can be generally divided into two main categories: protein-based and nucleic acid-based. Both can be obtained either by directly mining from natural genetic components or by engineering the existing genetic components for novel specificity or improved characteristics. A wide range of new technologies have enabled rapid engineering and discovery of new biosensors, which are paving the way for a new era of biotechnological progress. Here, we review recent advances in the design, optimization, and applications of in vivo biosensors in the field of metabolic engineering and synthetic biology.     

Progress in enzyme inhibition based detection of pesticides

The previous few decades have seen the development of biosensors and their use in monitoring of pesticides in food and environmental samples. Although inhibition‐based biosensors have been subject of several recent research works, their performance characteristics greatly depend on the type of immobilization and the presence of interfering compounds in the samples. Moreover, sensitivity, detection limits, and rapidity of the response are few of the other major features that need to be investigated further if they are to become operationally user‐friendly. This review will highlight research carried out in the past on biosensors that are based on enzyme inhibition for determination of organophosphorus compounds and carbamate pesticides.     

Live imaging of protein kinase activities in transgenic mice expressing FRET biosensors

Genetically-encoded biosensors based on the principle of F?rster resonance energy transfer (FRET) have been widely used in biology to visualize the spatiotemporal dynamics of signaling molecules. Despite the increasing multitude of these biosensors, their application has been mostly limited to cultured cells with transient biosensor expression, due to particular difficulties in the development of transgenic mice that express FRET biosensors. In this study, we report the efficient generation of transgenic mouse lines expressing heritable and functional biosensors for ERK and PKA. These transgenic mice were created by the cytoplasmic co-injection of Tol2 transposase mRNA and a circular plasmid harbouring Tol2 recombination sites. High expression of the biosensors in a wide range of cell types allowed us to screen newborn mice simply by inspection. Observation of these transgenic mice by two-photon excitation microscopy yielded real-time activity maps of ERK and PKA in various tissues, with greatly improved signal-to-background ratios. Our transgenic mice may be bred into diverse genetic backgrounds; moreover, the protocol we have developed paves the way for the generation of transgenic mice that express other FRET biosensors, with important applications in the characterization of physiological and pathological signal transduction events in addition to drug development and screening.     

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

Recent metagenomic studies have provided an unprecedented wealth of data, which are revolutionizing our understanding of virus diversity. A redrawn landscape highlights viruses as active players in the phytobiome, and surveys have uncovered their positive roles in environmental stress tolerance of plants. Viral infectious clones are key tools for functional characterization of known and newly identified viruses. Knowledge of viruses and their components has been instrumental for the development of modern plant molecular biology and biotechnology. In this review, we provide extensive guidelines built on current synthetic biology advances that streamline infectious clone assembly, thus lessening a major technical constraint of plant virology. The focus is on generation of infectious clones in binary T‐DNA vectors, which are delivered efficiently to plants by Agrobacterium. We then summarize recent applications of plant viruses and explore emerging trends in microbiology, bacterial and human virology that, once translated to plant virology, could lead to the development of virus‐based gene therapies for ad hoc engineering of plant traits. The systematic characterization of plant virus roles in the phytobiome and next‐generation virus‐based tools will be indispensable landmarks in the synthetic biology roadmap to better crops.     

Engineering and control of biological systems: A new way to tackle complex diseases

The ongoing merge between engineering and biology has contributed to the emerging field of synthetic biology. The defining features of this new discipline are abstraction and standardisation of biological parts, decoupling between parts to prevent undesired cross-talking, and the application of quantitative modelling of synthetic genetic circuits in order to guide their design. Most of the efforts in the field of synthetic biology in the last decade have been devoted to the design and development of functional gene circuits in prokaryotes and unicellular eukaryotes. Researchers have used synthetic biology not only to engineer new functions in the cell, but also to build simpler models of endogenous gene regulatory networks to gain knowledge of the "rules" governing their wiring diagram. However, the need for innovative approaches to study and modify complex signalling and regulatory networks in mammalian cells and multicellular organisms has prompted advances of synthetic biology also in these species, thus contributing to develop innovative ways to tackle human diseases. In this work, we will review the latest progress in synthetic biology and the most significant developments achieved so far, both in unicellular and multicellular organisms, with emphasis on human health.     

Modularized synthetic biology enabled intelligent biosensors

Biosensors that sense the concentration of a specified target and produce a specific signal output have become important technology for biological analysis. Recently, intelligent biosensors have received great interest due to their adaptability to meet sophisticated demands. Advances in developing standard modules and carriers in synthetic biology have shed light on intelligent biosensors that can implement advanced analytical processing to better accommodate practical applications. This review focuses on intelligent synthetic biology-enabled biosensors (SBBs). First, we illustrate recent progress in intelligent SBBs with the capability of computation, memory storage, and self-calibration. Then, we discuss emerging applications of SBBs in point-of-care testing (POCT) and wearable monitoring. Finally, future perspectives on intelligent SBBs are proposed.     

Label‐free indicator‐free nucleic acid biosensors using carbon nanotubes

Carbon nanotubes (CNTs) are promising components for electrical biosensors due to their high surface‐to‐volume ratio and improved electron transfer properties. This review surveys CNT‐based label‐free indicator‐free biosensing strategies that have been demonstrated for the sensitive detection of nucleic acids. After an introduction to CNTs, the fabrication of biosensors and techniques for the immobilization of probe nucleic acids are outlined. Subsequently, two major label‐free strategies namely electrochemical transduction and field‐effect detection are presented. The focus is on direct detection methods that avoid labels, indicators, intercalating agents, mediators, and even secondary receptors. The review concludes with a comparison between the various biosensors and presents ways of engineering them so that they can be deployed in realistic diagnostic applications.     

无细胞合成生物学：革新生物医药产业的新策略

无细胞合成生物系统,能够在体外完成生命转录翻译过程,因体系灵活开放、便于控制、表达周期短、高耐受性等特点,可表达细胞系统难以表达的蛋白质。随着无细胞生物传感和体系冻干技术的不断发展,其在医药健康领域的应用不断拓展。本文综述了无细胞合成生物学在按需生物医药合成和便携式医疗检测等医药健康领域的研究进展,该体系的进一步发展有潜力实现更复杂后修饰蛋白质药物的合成、可丰富无细胞生物传感器类型并提高其灵敏性。无细胞合成生物学作为新兴工程策略,未来必将更好地应用于高通量医药蛋白质筛选、新型病原体的检测等医药健康领域。     

DNA组装新方法的研究进展

2010年,蕈状支原体Mycoplasma mycoides的人工合成,迎来了合成生物学的崭新时代.这种突破性的进展主要得益于酵母自身强大的DNA体内重组能力.近几年来,除了利用体内重组的DNA大片段拼接技术,基于连接或聚合思想的不同尺度的DNA体外组装方法也相继出现,如Biobrick\Bglbrick、SLIC与Gibson等温一步法等,这些方法的应用加快了合成生物学功能元件库、生物合成途径乃至微生物染色体的人工构建.事实上,目前所建立的各种DNA组装方法,均是由DNA分子拼接理念(包括两分子衔接思想与多片段组装模式)衍生而来.文中将在介绍DNA组装基本理念的基础上,对体内、体外主要的DNA组装方法进行简要梳理,希望为不同类型的合成生物学功能器件及生物合成途径的构造提供参考与借鉴.     

酿酒酵母代谢工程技术

自20世纪90年代初期诞生以来,代谢工程历经了30年的快速发展。作为代谢工程的首选底盘细胞之一,酿酒酵母细胞工厂已被广泛应用于大量大宗化学品和新型高附加值生物活性物质的生物制造,在能源、医药和环境等领域取得了巨大的突破。近年来,合成生物学、生物信息学以及机器学习等相关技术也极大地促进了代谢工程的技术发展和应用。文中回顾了近30年来酿酒酵母代谢工程重要的技术发展,首先总结了经典代谢工程的常用方法和策略,以及在此基础上发展而来的系统代谢工程和合成生物学驱动的代谢工程技术。最后结合最新技术发展趋势,展望了未来酿酒酵母代谢工程发展的新方向。     

Fluorescent biosensors — Probing protein kinase function in cancer and drug discovery

One of the challenges of modern biology and medicine is to visualize biomolecules in their natural environment, in real-time and in a non-invasive fashion, so as to gain insight into their physiological behavior and highlight alterations in pathological settings, which will enable to devise appropriate therapeutic strategies. Fluorescent biosensors constitute a class of imaging agents which have provided major insights into the function and regulation of enzymes in their cellular context. GFP-based reporters and genetically-encoded FRET biosensors, have been successfully applied to study protein kinases in living cells with high spatial and temporal resolution. In parallel, combined efforts in fluorescence chemistry and in chemical biology have enabled the design of non-genetic, polypeptide biosensors coupled to small synthetic fluorescent probes, which have been applied to monitor protein kinase activities in vitro and in more complex biological samples, with an equally successful outcome. From a biomedical perspective, fluorescent biosensor technology is well suited to development of diagnostic approaches, for monitoring disease progression and for evaluating response to therapeutics. Moreover it constitutes an attractive technology for drug discovery programs, for high content, high throughput screening assays, to assess the potency of new hits and optimize lead compounds, whilst also serving to characterize drugs developed through rational design. This review describes the utility and versatility of fluorescence biosensor technology to probe protein kinases with a specific focus on CDK/cyclin biosensors we have developed to probe abundance, activity and conformation. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

How to make a minimal genome for synthetic minimal cell

As a key focus of synthetic biology, building a minimal artificial cell has given rise to many discussions. A synthetic minimal cell will provide an appropriate chassis to integrate functional synthetic parts, devices and systems with functions that cannot generally be found in nature. The design and construction of a functional minimal genome is a key step while building such a cell/chassis since all the cell functions can be traced back to the genome. Kinds of approaches, based on bioinformatics and molecular biology, have been developed and proceeded to derive essential genes and minimal gene sets for the synthetic minimal genome. Experiments about streamlining genomes of model bacteria revealed genome reduction led to unanticipated beneficial properties, such as high electroporation efficiency and accurate propagation of recombinant genes and plasmids that were unstable in other strains. Recent achievements in chemical synthesis technology for large DNA segments together with the rapid development of the whole-genome sequencing, have transferred synthesis of genes to assembly of the whole genomes based on oligonucleotides, and thus created strong preconditions for synthesis of artificial minimal genome. Here in this article, we review briefly the history and current state of research in this field and summarize the main methods for making a minimal genome. We also discuss the impacts of minimized genome on metabolism and regulation of artificial cell.     

历久弥新：进化中的代谢工程

20世纪90年代,Bailey及Stephanopoulos等提出了经典代谢工程的理念,旨在利用DNA重组技术对代谢网络进行改造,以达到细胞性能改善,目标产物增加的目的。自代谢工程诞生以来的30年,生命科学蓬勃发展,基因组学、系统生物学、合成生物学等新学科不断涌现,为代谢工程的发展注入了新的内涵与活力。经典代谢工程研究已进入到前所未有的系统代谢工程阶段。组学技术、基因组代谢模型、元件组装、回路设计、动态控制、基因组编辑等合成生物学工具与策略的应用,大大提升了复杂代谢的设计与合成能力;机器学习的介入以及进化工程与代谢工程的结合,为系统代谢工程的未来开辟了新的方向。文中对过去30年代谢工程的发展趋势作了梳理,介绍了代谢工程在发展中不断创新的理论与方法及其应用。     

不同处理方法对海稻米中γ-氨基丁酸提取率的影响

以海稻米为研究对象,研究提取温度、提取溶剂、料液比、提取时间、提取次数等5个因素对海稻米中γ 氨基丁酸（GABA）提取率的影响,采用正交试验分析方法确定海稻米中GABA最优工艺条件。结果表明：海稻米中GABA的最佳提取工艺为：提取溶剂为水、提取时间为1 h、提取次数3次、提取温度60 ℃、提取物料比1 g∶15 mL,在此提取条件下的提取率为6.2μg/g。     

Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways

Cells are filled with biosensors, molecular systems that measure the state of the cell and respond by regulating host processes. In much the same way that an engineer would monitor a chemical reactor, the cell uses these sensors to monitor changing intracellular environments and produce consistent behavior despite the variable environment. While natural systems derive a clear benefit from pathway regulation, past research efforts in engineering cellular metabolism have focused on introducing new pathways and removing existing pathway regulation. Synthetic biology is a rapidly growing field that focuses on the development of new tools that support the design, construction, and optimization of biological systems. Recent advances have been made in the design of genetically-encoded biosensors and the application of this class of molecular tools for optimizing and regulating heterologous pathways. Biosensors to cellular metabolites can be taken directly from natural systems, engineered from natural sensors, or constructed entirely in vitro. When linked to reporters, such as antibiotic resistance markers, these metabolite sensors can be used to report on pathway productivity, allowing high-throughput screening for pathway optimization. Future directions will focus on the application of biosensors to introduce feedback control into metabolic pathways, providing dynamic control strategies to increase the efficient use of cellular resources and pathway reliability.     

合成生物学方法构建电活性大肠杆菌

电活性微生物具有独特的在细胞内外环境之间传递电子的能力。在对天然电活性微生物电子传递机制充分研究的基础上,通过合成生物学方法异源构建天然电活性微生物电子传递结构基础也可以将遗传背景清晰的非电活性大肠杆菌改造为电活性微生物。构建获得的工程化电活性大肠杆菌可以直接应用于微生物燃料电池和生物传感器等领域,同时也可以作为底盘细胞整合相应的目标产物合成通路实现电能驱动的生物合成。本文以合成生物学方法构建电活性大肠杆菌为主题,详细阐述天然电活性微生物电子传递的机理及结构基础,总结了工程化电活性大肠杆菌的构建策略、成功案例以及应用领域,并对合成生物学方法构建电活性大肠杆菌未来的研究方向进行了展望。     

Microbial biosensors: a review

A microbial biosensor is an analytical device which integrates microorganism(s) with a physical transducer to generate a measurable signal proportional to the concentration of analytes. In recent years, a large number of microbial biosensors have been developed for environmental, food, and biomedical applications. Starting with the discussion of various sensing techniques commonly used in microbial biosensing, this review article concentrates on the summarization of the recent progress in the fabrication and application of microbial biosensors based on amperometry, potentiometry, conductometry, voltammetry, microbial fuel cell, fluorescence, bioluminescence, and colorimetry, respectively. Prospective strategies for the design of future microbial biosensors will also be discussed.     

A revolutionary tool: CRISPR technology plays an important role in construction of intelligentized gene circuits

With the development of synthetic biology, synthetic gene circuits have shown great applied potential in medicine, biology, and as commodity chemicals. An ultimate challenge in the construction of gene circuits is the lack of effective, programmable, secure and sequence‐specific gene editing tools. The clustered regularly interspaced short palindromic repeat (CRISPR) system, a CRISPR‐associated RNA‐guided endonuclease Cas9 (CRISPR‐associated protein 9)‐targeted genome editing tool, has recently been applied in engineering gene circuits for its unique properties‐operability, high efficiency and programmability. The traditional single‐targeted therapy cannot effectively distinguish tumour cells from normal cells, and gene therapy for single targets has poor anti‐tumour effects, which severely limits the application of gene therapy. Currently, the design of gene circuits using tumour‐specific targets based on CRISPR/Cas systems provides a new way for precision cancer therapy. Hence, the application of intelligentized gene circuits based on CRISPR technology effectively guarantees the safety, efficiency and specificity of cancer therapy. Here, we assessed the use of synthetic gene circuits and if the CRISPR system could be used, especially artificial switch‐inducible Cas9, to more effectively target and treat tumour cells. Moreover, we also discussed recent advances, prospectives and underlying challenges in CRISPR‐based gene circuit development.