scholarly journals <italic>In vitro</italic> synthetic biology: Cell-free protein synthesis

2017 ◽  
Vol 62 (33) ◽  
pp. 3851-3860
Author(s):  
Yang LIU ◽  
XiaoCui GUO ◽  
JinHui GENG ◽  
Yi JIAO ◽  
JinPeng HAN ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Synthetic Biology ◽  
Free Protein ◽  
Cell Free Protein Synthesis

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 Synthetic Biology Bicistronic Designs Support Gene Expression Equally Well in vitro and in vivo

2020 ◽  
Vol 17 (1) ◽  
pp. 13-20
Author(s):  
Owen Koucky ◽  
Jacob Wagner ◽  
Sofia Aguilera ◽  
Benjamin Bashaw ◽  
Queena Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Synthetic Biology ◽  
Protein Production ◽  
Persistent Problem ◽  
Free Protein ◽  
Microfluidic Droplets ◽  
Cell Free Protein Synthesis

Synthetic biology integrates molecular biology tools and an engineering mindset to address challenges in medicine, agriculture, bioremediation, and biomanufacturing. A persistent problem in synthetic biology has been designing genetic circuits that produce predictable levels of protein. In 2013, Mutalik and colleagues developed bicistronic designs (BCDs) that make protein production more predicable in bacterial cells (in vivo). With the growing interest in producing proteins outside of cells (in vitro), we wanted to know if BCDs would work as predictably in cell-free protein synthesis (CFPS) as they do in E. coli cells. We tested 20 BCDs in CFPS and found they performed very similarly in vitro and in vivo. As a step toward developing methods for protein production in artificial cells, we also tested 3 BCDs inside nanoliter-scaled microfluidic droplets. The BCDs worked well in the microfluidic droplets, but their relative protein production levels were not as predictable as expected. These results suggest that the conditions under which gene expression happens in droplets result in a different relationship between genetic control elements such as BCDs and protein production than exists in batch CFPS or in cells. KEYWORDS: Bicistronic Design; Synthetic Biology; Cell-Free Protein Synthesis; Microfluidics

scholarly journals Development and comparison of cell-free protein synthesis systems derived from typical bacterial chassis

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Liyuan Zhang ◽  
Xiaomei Lin ◽  
Ting Wang ◽  
Wei Guo ◽  
Yuan Lu
Keyword(s):  
Protein Synthesis ◽  
Protein Production ◽  
Influential Factors ◽  
Competent Cell ◽  
Free Protein ◽  
Application Range ◽  
E Coli ◽  
Short Time ◽  
Cell Free Protein Synthesis

AbstractCell-free protein synthesis (CFPS) systems have become an ideal choice for pathway prototyping, protein production, and biosensing, due to their high controllability, tolerance, stability, and ability to produce proteins in a short time. At present, the widely used CFPS systems are mainly based on Escherichia coli strain. Bacillus subtilis, Corynebacterium glutamate, and Vibrio natriegens are potential chassis cells for many biotechnological applications with their respective characteristics. Therefore, to expand the platform of the CFPS systems and options for protein production, four prokaryotes, E. coli, B. subtilis, C. glutamate, and V. natriegens were selected as host organisms to construct the CFPS systems and be compared. Moreover, the process parameters of the CFPS system were optimized, including the codon usage, plasmid synthesis competent cell selection, plasmid concentration, ribosomal binding site (RBS), and CFPS system reagent components. By optimizing and comparing the main influencing factors of different CFPS systems, the systems can be optimized directly for the most influential factors to further improve the protein yield of the systems. In addition, to demonstrate the applicability of the CFPS systems, it was proved that the four CFPS systems all had the potential to produce therapeutic proteins, and they could produce the receptor-binding domain (RBD) protein of SARS-CoV-2 with functional activity. They not only could expand the potential options for in vitro protein production, but also could increase the application range of the system by expanding the cell-free protein synthesis platform.

scholarly journals In Vitro Reconstruction of Nonribosomal Peptide Biosynthesis Directly from DNA Using Cell-Free Protein Synthesis

2016 ◽  
Vol 6 (1) ◽  
pp. 39-44 ◽  
Author(s):  
Anthony W. Goering ◽  
Jian Li ◽  
Ryan A. McClure ◽  
Regan J. Thomson ◽  
Michael C. Jewett ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Nonribosomal Peptide ◽  
Free Protein ◽  
Peptide Biosynthesis ◽  
Cell Free Protein Synthesis

scholarly journals Two fractions required for cell-free protein synthesis with components from rabbit reticulocytes

1969 ◽  
Vol 115 (3) ◽  
pp. 523-527 ◽  
Author(s):  
Brian B. Cohen
Keyword(s):  
Protein Synthesis ◽  
Active Component ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Analytical Ultracentrifuge ◽  
Active Components ◽  
Free Protein ◽  
Cell Free Protein Synthesis ◽  
Rabbit Reticulocytes

An extract was prepared from rabbit reticulocyte ribosomes after treatment with potassium chloride as described by Miller, Hamada, Yang, Cohen & Schweet (1967). This extract has been shown to convert monoribosomes into polyribosomes during protein synthesis in vitro (Cohen, 1968). The nature of this extract was studied in greater detail. Centrifugation of the extract through a sucrose density gradient separated the activity into a fast-sedimenting fraction. The two fractions were shown to have different functions in stimulating cell-free protein synthesis and their active components were shown to be protein or partly protein in nature. Each fraction was analysed by electrophoresis and in the analytical ultracentrifuge. It was concluded that the active component in the fast-sedimenting fraction had a sedimentation coefficient of 15·5s and that of the slow-sedimenting fraction 10·5s.

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