Synthetic therapeutic gene circuits in mammalian cells

In the emerging field of synthetic biology, scientists are focusing on designing and creating functional devices, systems, and organisms with novel functions by engineering and assembling standardised biological building blocks. The progress of synthetic biology has significantly advanced the design of functional gene networks that can reprogram metabolic activities in mammalian cells and provide new therapeutic opportunities for future gene- and cell-based therapies. In this review, we describe the most recent advances in synthetic mammalian gene networks designed for biomedical applications, including how these synthetic therapeutic gene circuits can be assembled to control signalling networks and applied to treat metabolic disorders, cancer, and immune diseases. We conclude by discussing the various challenges and future prospects of using synthetic mammalian gene networks for disease therapy.     

Synthetic genetic circuits for programmable biological functionalities

Living organisms evolve complex genetic networks to interact with the environment. Due to the rapid development of synthetic biology, various modularized genetic parts and units have been identified from these networks. They have been employed to construct synthetic genetic circuits, including toggle switches, oscillators, feedback loops and Boolean logic gates. Building on these circuits, complex genetic machines with capabilities in programmable decision-making could be created. Consequently, these accomplishments have led to novel applications, such as dynamic and autonomous modulation of metabolic networks, directed evolution of biological units, remote and targeted diagnostics and therapies, as well as biological containment methods to prevent release of engineered microorganisms and genetic materials. Herein, we outline the principles in genetic circuit design that have initiated a new chapter in transforming concepts to realistic applications. The features of modularized building blocks and circuit architecture that facilitate realization of circuits for a variety of novel applications are discussed. Furthermore, recent advances and challenges in employing genetic circuits to impart microorganisms with distinct and programmable functionalities are highlighted. We envision that this review gives new insights into the design of synthetic genetic circuits and offers a guideline for the implementation of different circuits in various aspects of biotechnology and bioengineering.     

Programming microbial population dynamics by engineered cell-cell communication

A major aim of synthetic biology is to program novel cellular behavior using engineered gene circuits. Early endeavors focused on building simple circuits that fulfill simple functions, such as logic gates, bistable toggle switches, and oscillators. These gene circuits have primarily focused on single-cell behaviors since they operate intracellularly. Thus, they are often susceptible to cell-cell variations due to stochastic gene expression. Cell-cell communication offers an efficient strategy to coordinate cellular behavior at the population level. To this end, we review recent advances in engineering cell-cell communication to achieve reliable population dynamics, spanning from communication within single species to multispecies, from one-way sender-receiver communication to two-way communication in synthetic microbial ecosystems. These engineered systems serve as well-defined model systems to better understand design principles of their naturally occurring counterparts and to facilitate novel biotechnology applications.     

The emerging age of cell-free synthetic biology

The engineering of and mastery over biological parts has catalyzed the emergence of synthetic biology. This field has grown exponentially in the past decade. As increasingly more applications of synthetic biology are pursued, more challenges are encountered, such as delivering genetic material into cells and optimizing genetic circuits in vivo. An in vitro or cell-free approach to synthetic biology simplifies and avoids many of the pitfalls of in vivo synthetic biology. In this review, we describe some of the innate features that make cell-free systems compelling platforms for synthetic biology and discuss emerging improvements of cell-free technologies. We also select and highlight recent and emerging applications of cell-free synthetic biology.     

Forward engineering of synthetic bio-logical AND gates

The field of synthetic biology has produced genetic circuits capable of emulating functional paradigms seen in digital electronic circuits. Examples are bistable switches, oscillators, and logic gates. The present work combines detailed mechanistic-kinetic models and stochastic simulation techniques as well as the techniques of in vivo molecular biology to study the potential of a synthetic, single promoter AND gate. This device is composed of elements of the tet, lac, and λ-phage promoters and is responsive to the commonly used inducers IPTG and aTc, producing GFP as an output signal. The quantitative behavior of the AND gate phenotype is studied both in numero and in vivo as a function of promoter topology. The model is constructed from kinetic data obtained from the literature and yields clearly defined ON/OFF logical behavior at realistic inducer concentrations. These behaviors are matched with observed in vivo data obtained through fluorescence-activated cell sorting. The effect of incomplete repression by weaker LacI repressor is also investigated and quantified. The simulation results, coupled with in vivo data, not only identify important design degrees of freedom, but also provide parameters that can be used to guide future synthetic designs using these common regulatory elements.     

Engineering light-gated ion channels

Ion channels are gated by a variety of stimuli, including ligands, voltage, membrane tension, temperature, and even light. Natural gates can be altered and augmented using synthetic chemistry and molecular biology to develop channels with completely new functional properties. Light-sensitive channels are particularly attractive because optical manipulation offers a high degree of spatial and temporal control. Over the last few decades, several channels have been successfully rendered responsive to light, including the nicotinic acetylcholine receptor, gramicidin A, a voltage-gated potassium channel, an ionotropic glutamate receptor, alpha-hemolysin, and a mechanosensitive channel. Very recently, naturally occurring light-gated cation channels have been discovered. This review covers the molecular principles that guide the engineering of light-gated ion channels for applications in biology and medicine.     

Multi-Input Distributed Classifiers for Synthetic Genetic Circuits

For practical construction of complex synthetic genetic networks able to perform elaborate functions it is important to have a pool of relatively simple modules with different functionality which can be compounded together. To complement engineering of very different existing synthetic genetic devices such as switches, oscillators or logical gates, we propose and develop here a design of synthetic multi-input classifier based on a recently introduced distributed classifier concept. A heterogeneous population of cells acts as a single classifier, whose output is obtained by summarizing the outputs of individual cells. The learning ability is achieved by pruning the population, instead of tuning parameters of an individual cell. The present paper is focused on evaluating two possible schemes of multi-input gene classifier circuits. We demonstrate their suitability for implementing a multi-input distributed classifier capable of separating data which are inseparable for single-input classifiers, and characterize performance of the classifiers by analytical and numerical results. The simpler scheme implements a linear classifier in a single cell and is targeted at separable classification problems with simple class borders. A hard learning strategy is used to train a distributed classifier by removing from the population any cell answering incorrectly to at least one training example. The other scheme implements a circuit with a bell-shaped response in a single cell to allow potentially arbitrary shape of the classification border in the input space of a distributed classifier. Inseparable classification problems are addressed using soft learning strategy, characterized by probabilistic decision to keep or discard a cell at each training iteration. We expect that our classifier design contributes to the development of robust and predictable synthetic biosensors, which have the potential to affect applications in a lot of fields, including that of medicine and industry.     

杆状病毒介导的基因传递逃避补体攻击的措施

血清补体系统是杆状病毒介导基因传递过程中的主要抑制因素.概述逃避补体攻击的有效措施:选择免疫赦免组织作为杆状病毒介导基因传递的靶标,例如眼睛、脑和睾丸;体外转导也能有效地逃避补体的攻击;利用补体的药理抑制剂或者对杆状病毒载体进行加工修饰,例如聚合物包被、假型化和展示补体抑制剂等,可以消除杆状病毒对补体的敏感性.有效地逃避补体攻击有助于杆状病毒广泛应用于基因治疗.     

Archaeal proteasomes: potential in metabolic engineering

Archaea are a valuable source of enzymes for industrial and scientific applications because of their ability to survive extreme conditions including high salt and temperature. Thanks to advances in molecular biology and genetics, archaea are also attractive hosts for metabolic engineering. Understanding how energy-dependent proteases and chaperones function to maintain protein quality control is key to high-level synthesis of recombinant products. In archaea, proteasomes are central players in energy-dependent proteolysis and form elaborate nanocompartments that degrade proteins into oligopeptides by processive hydrolysis. The catalytic core responsible for this proteolytic activity is the 20S proteasome, a barrel-shaped particle with a central channel and axial gates on each end that limit substrate access to a central proteolytic chamber. AAA proteins (ATPases associated with various cellular activities) are likely to play several roles in mediating energy-dependent proteolysis by the proteasome. These include ATP binding/hydrolysis, substrate binding/unfolding, opening of the axial gates, and translocation of substrate into the proteolytic chamber.     

Design and analysis of a robust genetic Muller C-element

This paper presents results on the design and analysis of a robust genetic Muller C-element. The Muller C-element is a standard logic gate commonly used to synchronize independent processes in most asynchronous electronic circuits. Synthetic biological logic gates have been previously demonstrated, but there remain many open issues in the design of sequential (state-holding) logic operations. Three designs are considered for the genetic Muller C-element: a majority gate, a toggle switch, and a speed-independent implementation. While the three designs are logically equivalent, each design requires different assumptions to operate correctly. The majority gate design requires the most timing assumptions, the speed-independent design requires the least, and the toggle switch design is a compromise between the two. This paper examines the robustness of these designs as well as the effects of parameter variation using stochastic simulation. The results show that robustness to timing assumptions does not necessarily increase reliability, suggesting that modifications to existing logic design tools are going to be necessary for synthetic biology. Parameter variation simulations yield further insights into the design principles necessary for building robust genetic gates. The results suggest that high gene count, cooperativity of at least two, tight repression, and balanced decay rates are necessary for robust gates. Finally, this paper presents a potential application of the genetic Muller C-element as a quorum-mediated trigger.     

Rapid and orthogonal logic gating with a gibberellin-induced dimerization system

Using a newly synthesized gibberellin analog containing an acetoxymethyl group (GA(3)-AM) and its binding proteins, we developed an efficient chemically inducible dimerization (CID) system that is completely orthogonal to existing rapamycin-mediated protein dimerization. Combining the two systems should allow applications that have been difficult or impossible with only one CID system. By using both chemical inputs (rapamycin and GA(3)-AM), we designed and synthesized Boolean logic gates in living mammalian cells. These gates produced output signals such as fluorescence and membrane ruffling on a timescale of seconds, substantially faster than earlier intracellular logic gates. The use of two orthogonal dimerization systems in the same cell also allows for finer modulation of protein perturbations than is possible with a single dimerizer.     

面向先进生物燃料的合成生物学

先进生物燃料一般指来自于非粮食原料的交通运输用生物燃料。近年来,先进生物燃料的发展引起了众多国家的浓厚兴趣,然而,先进生物燃料正处于关键的技术研发阶段,还需经过大量研发以突破技术障碍和示范生产活动后方能进行商业化部署。过去10年内,合成生物学研究大量兴起并不断取得突破,使人们有可能人工设计构建新的高效生命系统,克服生物燃料发展的技术瓶颈,进行先进生物燃料的生产。在介绍先进生物燃料与合成生物学的发展现状的基础上,分析了合成生物学在先进生物燃料研发中的重要价值与研发进展,探讨了合成生物学的发展潜力。