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引用次数: 0
摘要
自我再生是生命系统的一项关键功能,需要在体外重现,以创建一个活的合成细胞。PURE 无细胞转录-翻译系统中蛋白质自我再生的一个主要限制因素是蛋白质浓度过高,远远超过了系统的蛋白质合成率。在这里,我们能够将非核糖体 PURE 蛋白浓度大幅降低至 97.3%,同时提高蛋白质合成效率。虽然拥挤剂在最初的 PURE 配方中不起作用,但我们发现,在高度稀释的 PURE 配方中,添加 6% 的葡聚糖可大大提高蛋白质合成率和总蛋白质产量。这些新的 PURE 配方将在许多无细胞合成生物学应用中发挥作用,我们估计 PURE 现在可以支持所有 36 种非核糖体蛋白的完全自我再生,这是向开发通用生化构建器和活体合成细胞迈出的关键一步。
Towards Self-regeneration: Exploring the Limits of Protein Synthesis in the Protein Synthesis Using Recombinant Elements (PURE) Cell-free Transcription-Translation System.
Self-regeneration is a key function of living systems that needs to be recapitulated in vitro to create a living synthetic cell. A major limiting factor for protein self-regeneration in the PURE cell-free transcription-translation system is its high protein concentration, which far exceeds the system's protein synthesis rate. Here, we were able to drastically reduce the nonribosomal PURE protein concentration up to 97.3% while increasing protein synthesis efficiency. Although crowding agents were not effective in the original PURE formulation, we found that in highly dilute PURE formulations, addition of 6% dextran considerably increased protein synthesis rate and total protein yield. These new PURE formulations will be useful for many cell-free synthetic biology applications, and we estimate that PURE can now support the complete self-regeneration of all 36 nonribosomal proteins, which is a critical step toward the development of a universal biochemical constructor and living synthetic cell.
期刊介绍:
The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism.
Topics may include, but are not limited to:
Design and optimization of genetic systems
Genetic circuit design and their principles for their organization into programs
Computational methods to aid the design of genetic systems
Experimental methods to quantify genetic parts, circuits, and metabolic fluxes
Genetic parts libraries: their creation, analysis, and ontological representation
Protein engineering including computational design
Metabolic engineering and cellular manufacturing, including biomass conversion
Natural product access, engineering, and production
Creative and innovative applications of cellular programming
Medical applications, tissue engineering, and the programming of therapeutic cells
Minimal cell design and construction
Genomics and genome replacement strategies
Viral engineering
Automated and robotic assembly platforms for synthetic biology
DNA synthesis methodologies
Metagenomics and synthetic metagenomic analysis
Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction
Gene optimization
Methods for genome-scale measurements of transcription and metabolomics
Systems biology and methods to integrate multiple data sources
in vitro and cell-free synthetic biology and molecular programming
Nucleic acid engineering.