Cell-free biology: exploiting the interface between synthetic biology and synthetic chemistry

Harris, Dylan; Jewett, Michael C.

doi:10.1016/j.copbio.2012.02.002

Cited by 56 publications

(42 citation statements)

References 59 publications

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“…One potential route for the further development of S. venezuelae and the characterization of its genetic parts is the introduction of an in vitro transcription-translation system (TX-TL). TX-TL has recently been developed as a highly adaptable tool for bottom-up synthetic biology and is based on a whole-cell extract [6][7][8][9] to synthesize recombinant proteins from the chemical building blocks of life.…”

Section: Introductionmentioning

confidence: 99%

Streptomyces venezuelae TX‐TL – a next generation cell‐free synthetic biology tool

Moore

Lai

Needham

et al. 2017

Biotechnology Journal

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Streptomyces venezuelae is a promising chassis in synthetic biology for fine chemical and secondary metabolite pathway engineering. The potential of S. venezuelae could be further realized by expanding its capability with the introduction of its own in vitro transcription-translation (TX-TL) system. TX-TL is a fast and expanding technology for bottom-up design of complex gene expression tools, biosensors and protein manufacturing. Herein, we introduce a S. venezuelae TX-TL platform by reporting a streamlined protocol for cell-extract preparation, demonstrating high-yield synthesis of a codon-optimized sfGFP reporter and the prototyping of a synthetic tetracycline-inducible promoter in S. venezuelae TX-TL based on the tetO-TetR repressor system. The aim of this system is to provide a host for the homologous production of exotic enzymes from Actinobacteria secondary metabolism in vitro. As an example, the authors demonstrate the soluble synthesis of a selection of enzymes (12-70 kDa) from the Streptomyces rimosus oxytetracycline pathway.

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Section: Introductionmentioning

confidence: 99%

Streptomyces venezuelae TX‐TL – a next generation cell‐free synthetic biology tool

Moore

Lai

Needham

et al. 2017

Biotechnology Journal

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“…These systems offer bottom-up approaches to investigate gene expression under minimal conditions without complication of cell division, host networks, and cellular organelles. [98][99][100] In addition, they establish a foundation toward using artificial cells for in vitro evolution of cellular components. 101,102 On the one hand, the development of artificial cells could directly benefit from the characterization of gene circuits in cell-free systems.…”

Section: The Information: the Study Of Genetic Modules Using Artificimentioning

confidence: 99%

“…On the other hand, the same pipeline of measuring gene circuit dynamics using cell-free systems could lead to fast-and high-throughput platforms for the design of synthetic circuits and components. 98,99 Indeed, synthetic expression systems are being established as ex vivo systems (outside natural and artificial cells) for fast characterization of synthetic genetic parts, including fluorescent proteins, 103 RBSs, 104 and hybrid DNA promoters. Using PURE system, 17 different fluorescent proteins were synthesized, screened, and quantified for their fluorescence intensities.…”

Section: The Information: the Study Of Genetic Modules Using Artificimentioning

confidence: 99%

The engineering of artificial cellular nanosystems using synthetic biology approaches

Tan

2014

WIREs Nanomed Nanobiotechnol

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Artificial cellular systems are minimal systems that mimic certain properties of natural cells, including signaling pathways, membranes, and metabolic pathways. These artificial cells (or protocells) can be constructed following a synthetic biology approach by assembling biomembranes, synthetic gene circuits, and cell-free expression systems. As artificial cells are built from bottom-up using minimal and a defined number of components, they are more amenable to predictive mathematical modeling and engineered controls when compared with natural cells. Indeed, artificial cells have been implemented as drug delivery machineries and in situ protein expression systems. Furthermore, artificial cells have been used as biomimetic systems to unveil new insights into functions of natural cells, which are otherwise difficult to investigate owing to their inherent complexity. It is our vision that the development of artificial cells would bring forth parallel advancements in synthetic biology, cell-free systems, and in vitro systems biology. For further resources related to this article, please visit the WIREs website. Conflict of interests: The authors declare that they have no competing financial interests.

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“…Without the limitations of cell walls or membranes, precise modulation of protein expression may be achieved by addition of exogenous factors, such as chaperones, isomerases, or posttranslationalmodifying enzymes, to manipulate translation and folding. Furthermore, these systems eliminate cell viability constraints and allow more efficient use of reactor volume (69). Of particular note, the Protein synthesis Using Recombinant Elements (PURE) system, which is a reconstituted system of highly purified components, provides exquisite control for adding or subtracting components tailored specifically to production of the protein or peptide of interest.…”

Section: Manufacturing Processesmentioning

confidence: 99%

Early Engineering Approaches to Improve Peptide Developability and Manufacturability

Furman

Chiu

Hunter

2014

AAPS J

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Abstract. Downstream success in Pharmaceutical Development requires thoughtful molecule design early in the lifetime of any potential therapeutic. Most therapeutic monoclonal antibodies are quite similar with respect to their developability properties. However, the properties of therapeutic peptides tend to be as diverse as the molecules themselves. Analysis of the primary sequence reveals sites of potential adverse posttranslational modifications including asparagine deamidation, aspartic acid isomerization, methionine, tryptophan, and cysteine oxidation and, potentially, chemical and proteolytic degradation liabilities that can impact the developability and manufacturability of a potential therapeutic peptide. Assessing these liabilities, both biophysically and functionally, early in a molecule's lifetime can drive a more effective path forward in the drug discovery process. In addition to these potential liabilities, more complex peptides that contain multiple disulfide bonds can pose particular challenges with respect to production and manufacturability. Approaches to reducing the disulfide bond complexity of these peptides are often explored with mixed success. Proteolytic degradation is a major contributor to decreased half-life and efficacy. Addressing this aspect of peptide stability early in the discovery process increases downstream success. We will address aspects of peptide sequence analysis, molecule complexity, developability analysis, and manufacturing routes that drive the decision making processes during peptide therapeutic development.

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Cell-free biology: exploiting the interface between synthetic biology and synthetic chemistry

Cited by 56 publications

References 59 publications

Streptomyces venezuelae TX‐TL – a next generation cell‐free synthetic biology tool

Streptomyces venezuelae TX‐TL – a next generation cell‐free synthetic biology tool

The engineering of artificial cellular nanosystems using synthetic biology approaches

Early Engineering Approaches to Improve Peptide Developability and Manufacturability

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