Genetically Encoded Control of In Vitro Transcription-Translation Coupled DNA Replication.

IF 3.9 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2025-09-19 DOI:10.1021/acssynbio.5c00477

Sebastian Barthel, Maximilian Hoffmann-Becking, Islomjon G Karimov, Tobias J Erb

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引用次数: 0

Abstract

The bottom-up reconstruction of cellular functions has gained increasing attention for studying biological complexity and for developing advanced biotechnological tools, including synthetic cells. A fundamental challenge is the ability to control and replicate DNA-encoded information within basic in vitro transcription-translation (IVTT) systems. Here, we constructed a transcription-translation coupled DNA replication (TTcDR) system that is based on a modified PURE (Protein synthesis Using Recombinant Elements) IVTT system and Φ29 DNA polymerase, which is controlled by external signals. To this end, we first established and characterized a PUREfrex 1.0-based TTcDR system. We then constructed and optimized TetR-based control of TTcDR activity, either by DNA-encoded TetR or by supplying purified TetR. Our final DNA-encoded TetR circuit allows ∼1000-fold DNA replication, ∼100-fold repression, and ∼4-fold induction with anhydrotetracycline. Our results demonstrate the potential and challenges of controlling in vitro DNA replication, for example, for the evolution of in vitro systems.

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体外转录-翻译耦合DNA复制的遗传编码控制。

细胞功能的自底向上重建在研究生物复杂性和开发包括合成细胞在内的先进生物技术工具方面受到越来越多的关注。一个基本的挑战是控制和复制基本体外转录-翻译（IVTT）系统中dna编码信息的能力。在此，我们构建了一个转录-翻译耦合DNA复制（TTcDR）系统，该系统基于一个改进的PURE (Protein synthesis Using Recombinant Elements) IVTT系统和Φ29 DNA聚合酶，由外部信号控制。为此，我们首先建立并描述了一个基于PUREfrex 1.0的TTcDR系统。然后，我们构建并优化了基于TetR的TTcDR活性控制，通过dna编码的TetR或提供纯化的TetR。我们最终的DNA编码的TetR电路允许~ 1000倍的DNA复制，~ 100倍的抑制和~ 4倍的无水四环素诱导。我们的结果证明了控制体外DNA复制的潜力和挑战，例如，用于体外系统的进化。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

ACS Synthetic Biology 生物-

CiteScore

8.00

自引率

10.60%

发文量

380

审稿时长

6-12 weeks

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.