scholarly journals Cell-free synthetic biology for in vitro prototype engineering

2017 ◽  
Vol 45 (3) ◽  
pp. 785-791 ◽  
Author(s):  
Simon J. Moore ◽  
James T. MacDonald ◽  
Paul S. Freemont
Keyword(s):  
Synthetic Biology ◽  
Mathematical Modelling ◽  
Living Cells ◽  
Genetic Circuits ◽  
Precision Engineering ◽  
Medical Test ◽  
Iterative Design ◽  
Synthetic Life ◽  
Site Identification

Cell-free transcription–translation is an expanding field in synthetic biology as a rapid prototyping platform for blueprinting the design of synthetic biological devices. Exemplar efforts include translation of prototype designs into medical test kits for on-site identification of viruses (Zika and Ebola), while gene circuit cascades can be tested, debugged and re-designed within rapid turnover times. Coupled with mathematical modelling, this discipline lends itself towards the precision engineering of new synthetic life. The next stages of cell-free look set to unlock new microbial hosts that remain slow to engineer and unsuited to rapid iterative design cycles. It is hoped that the development of such systems will provide new tools to aid the transition from cell-free prototype designs to functioning synthetic genetic circuits and engineered natural product pathways in living cells.

scholarly journals Regulating synthetic gene networks in 3D materials

2012 ◽  
Vol 109 (38) ◽  
pp. 15217-15222 ◽  
Author(s):  
Tara L. Deans ◽  
Anirudha Singh ◽  
Matthew Gibson ◽  
Jennifer H. Elisseeff
Keyword(s):  
Synthetic Biology ◽  
Gene Networks ◽  
Materials Science ◽  
Genetic Circuits ◽  
Therapeutic Applications ◽  
Regulatory Processes ◽  
Cellular Events ◽  
The Matrix

Combining synthetic biology and materials science will enable more advanced studies of cellular regulatory processes, in addition to facilitating therapeutic applications of engineered gene networks. One approach is to couple genetic inducers into biomaterials, thereby generating 3D microenvironments that are capable of controlling intrinsic and extrinsic cellular events. Here, we have engineered biomaterials to present the genetic inducer, IPTG, with different modes of activating genetic circuits in vitro and in vivo. Gene circuits were activated in materials with IPTG embedded within the scaffold walls or chemically linked to the matrix. In addition, systemic applications of IPTG were used to induce genetic circuits in cells encapsulated into materials and implanted in vivo. The flexibility of modifying biomaterials with genetic inducers allows for patterned placement of these inducers that can be used to generate distinct patterns of gene expression. Together, these genetically interactive materials can be used to characterize genetic circuits in environments that more closely mimic cells’ natural 3D settings, to better explore complex cell–matrix and cell–cell interactions, and to facilitate therapeutic applications of synthetic biology.

scholarly journals Synthetic Life with Alternative Nucleic Acids as Genetic Materials

Molecules ◽  
2020 ◽  
Vol 25 (15) ◽  
pp. 3483
Author(s):  
Peng Nie ◽  
Yanfen Bai ◽  
Hui Mei
Keyword(s):  
Nucleic Acids ◽  
Genetic Information ◽  
Structural Parameters ◽  
Living Cells ◽  
Information Storage ◽  
Chemically Modified ◽  
Synthetic Life ◽  
Living Organisms

DNA, the fundamental genetic polymer of all living organisms on Earth, can be chemically modified to embrace novel functions that do not exist in nature. The key chemical and structural parameters for genetic information storage, heredity, and evolution have been elucidated, and many xenobiotic nucleic acids (XNAs) with non-canonical structures are developed as alternative genetic materials in vitro. However, it is still particularly challenging to replace DNAs with XNAs in living cells. This review outlines some recent studies in which the storage and propagation of genetic information are achieved in vivo by expanding genetic systems with XNAs.

scholarly journals A Fluorescent Split Aptamer for Visualizing RNA-RNA Assembly In Vivo

2017 ◽  
Author(s):  
Khalid K. Alam ◽  
Kwaku D. Tawiah ◽  
Matthew F. Lichte ◽  
David Porciani ◽  
Donald H. Burke
Keyword(s):  
Self Assembly ◽  
Living Cells ◽  
Rna Aptamers ◽  
Genetic Circuits ◽  
Fluorescent Reporter ◽  
Indirect Measures ◽  
Reporter Proteins ◽  
Circuit Function

AbstractRNA-RNA assembly governs key biological processes and is a powerful tool for engineering synthetic genetic circuits. Characterizing RNA assembly in living cells often involves monitoring fluorescent reporter proteins, which are at best indirect measures of underlying RNA-RNA hybridization events and are subject to additional temporal and load constraints associated with translation and activation of reporter proteins. In contrast, RNA aptamers that sequester small molecule dyes and activate their fluorescence are increasingly utilized in genetically-encoded strategies to report on RNA-level events. Split-aptamer systems have been rationally designed to generate signal upon hybridization of two or more discrete RNA transcripts, but none directly function when expressed in vivo. We reasoned that the improved physiological properties of the Broccoli aptamer enable construction of a split-aptamer system that could function in living cells. Here we present the Split-Broccoli system, in which self-assembly is nucleated by a thermostable, three-way junction RNA architecture and fluorescence activation requires both strands. Functional assembly of the system approximately follows second order kinetics in vitro and improves when cotranscribed, rather than when assembled from purified components. Split-Broccoli fluorescence is digital in vivo and retains functional modularity when fused to RNAs that regulate circuit function through RNA-RNA hybridization, as demonstrated with an RNA Toehold switch. Split-Broccoli represents the first functional split-aptamer system to operate in vivo. It offers a genetically-encoded and nondestructive platform to monitor and exploit RNA-RNA hybridization, whether as an all-RNA, stand-alone AND gate or as a tool for monitoring assembly of RNA-RNA hybrids.

scholarly journals Vida fabricada

2019 ◽  
Author(s):  
Manuel Porcar ◽  
Juli Peretó
Keyword(s):  
Synthetic Biology ◽  
Life Forms ◽  
Living Cells ◽  
Economic Benefits ◽  
Social Implications ◽  
Biological Complexity ◽  
The Social ◽  
Synthetic Life ◽  
Design And Manufacture ◽  
Rule Out

Since biology became secularised and the molecular scrutiny of life began, the possibility of artificially synthesising living cells in a laboratory became a tangible possibility. Contemporary synthetic biology aspires to design and manufacture new forms of life to obtain social and economic benefits. However, we cannot rule out the possibility that the creation of synthetic life forms may also bring scientific rewards in terms of a greater understanding of biological complexity, which we would not be able to access through analytical means. It is clear, therefore, that the term synthetic biology raises expectations, but it is no less true that it also causes concern. This article starts with a critique of the identification of cells as machines and discusses the current scope of synthetic biology and efforts to standardise it. We also outline some of the social implications of attempts to manufacture life.

scholarly journals “Cell-Free Synthetic Biology”: Synthetic Biology Meets Cell-Free Protein Synthesis

2019 ◽  
Vol 2 (4) ◽  
pp. 80
Author(s):  
Hong
Keyword(s):  
Protein Synthesis ◽  
Synthetic Biology ◽  
Biological Networks ◽  
Unnatural Amino Acids ◽  
Genetic Circuits ◽  
Free Protein ◽  
Protein Libraries ◽  
Cell Free Protein Synthesis

Since Nirenberg and Matthaei used cell-free protein synthesis (CFPS) to elucidate the genetic code in the early 1960s [1], the technology has been developed over the course of decades and applied to studying both fundamental and applied biology [2]. Cell-free synthetic biology integrating CFPS with synthetic biology has received attention as a powerful and rapid approach to characterize and engineer natural biological systems. The open nature of cell-free (or in vitro) biological platforms compared to in vivo systems brings an unprecedented level of control and freedom in design [3]. This versatile engineering toolkit has been used for debugging biological networks, constructing artificial cells, screening protein libraries, prototyping genetic circuits, developing biosensors, producing metabolites, and synthesizing complex proteins including antibodies, toxic proteins, membrane proteins, and novel proteins containing nonstandard (unnatural) amino acids. The Methods and Protocols “Cell-Free Synthetic Biology” Special Issue consists of a series of reviews, protocols, benchmarks, and research articles describing the current development and applications of cell-free synthetic biology in diverse areas. [...]

scholarly journals A Modular Computational Framework for the Design of Multicellular Genetic Circuits of Morphogenesis

2019 ◽  
Author(s):  
Calvin Lam ◽  
Leonardo Morsut
Keyword(s):  
Synthetic Biology ◽  
Mammalian Cells ◽  
Computational Models ◽  
Developmental Trajectories ◽  
Critical Parameters ◽  
Successful Implementation ◽  
Genetic Circuits ◽  
Computational Framework ◽  
Cell Cell

SUMMARYSynthetic development is a nascent field of research that uses the tools of synthetic biology to design genetic programs directing cellular patterning and morphogenesis in higher eukaryotic cells, such as mammalian cells. Synthetic genetic networks comprising cell-cell communications and morphogenesis effectors (e.g. adhesion) are generated and integrated into a cellular genome. Current design methods for these genetic programs rely on trial and error, which limit the number of possible circuits and parameter ranges that can be explored. By contrast, computational models act as rapid testing platforms, revealing the networks, signals, and responses required for achieving robust target structures. Here we introduce a computational framework, based on cellular Potts, where contact dependent cell-cell signaling networks and cellular responses can be chosen in a modular fashion. We represent and tune a number of recently described synthetic morphogenic trajectories in silico, such as those resulting in multilayered synthetic spheroids. Our parameters were tuned using a comparison with published in vitro experimental results. Our tuned parameters were then used to design and explore novel developmental trajectories for the formation of elongated and oscillatory structures. Here, multiple rounds of optimization suggested critical parameters for the successful implementation of these trajectories. The framework that we develop here could function as a testing ground to explore how synthetic biology tools can be used to create particular multicellular trajectories, as well as for understanding both imagined and extant developmental trajectories.

Carbon dots derived from Fusobacterium nucleatum for intracellular determination of Fe3+ and bioimaging both in vitro and in vivo

2021 ◽  
Author(s):  
Lijuan Liu ◽  
Shengting Zhang ◽  
Xiaodan Zheng ◽  
Hongmei Li ◽  
Qi Chen ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Fusobacterium Nucleatum ◽  
Living Cells ◽  
First Time ◽  
Fluorescent Carbon Dots ◽  
Fluorescent Carbon

Fusobacterium nucleatum has been employed for the first time to synthesize fluorescent carbon dots which could be applied for the determination of Fe3+ ions in living cells and bioimaging in vitro and in vivo with excellent biocompatibility.

Quinolimide-based peptide biosensor for probing p25 in vitro and in living cells

2021 ◽  
pp. 129929
Author(s):  
Francisco Fueyo-González ◽  
Rosario Herranz ◽  
Simona Plesselova ◽  
Maria D. Giron ◽  
Rafael Salto ◽  
...  
Keyword(s):  
Living Cells

scholarly journals CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Stefano Vecchione ◽  
Georg Fritz
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Integrated Circuits ◽  
High Efficiency ◽  
Genetic Circuit ◽  
Dna Assembly ◽  
Chromosomal Integration ◽  
Golden Gate ◽  
Genetic Circuits ◽  
E Coli

Abstract Background Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

Sign in / Sign up

Export Citation Format

Share Document