Preventing Multimer Formation in Commonly Used Synthetic Biology Plasmids.

IF 3.9 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2025-04-18 Epub Date: 2025-03-18 DOI:10.1021/acssynbio.4c00508

Elizabeth Vaisbourd, Anat Bren, Uri Alon, David S Glass

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Abstract

Plasmids are an essential tool for basic research and biotechnology applications. To optimize plasmid-based circuits, it is crucial to control plasmid integrity, including the formation of plasmid multimers. Multimers are tandem repeats of entire plasmids formed by failed dimer resolution during replication. Multimers can affect the behavior of synthetic circuits, especially ones that include DNA-editing enzymes. However, occurrence of multimers is not commonly assayed. Here we survey four commonly used plasmid backbones for occurrence of multimers in cloning (JM109) and wild-type (MG1655) strains of Escherichia coli. We find that multimers occur appreciably only in MG1655, with the fraction of plasmids existing as multimers increasing with both plasmid copy number and culture passaging. In contrast, transforming multimers into JM109 can yield strains that contain no singlet plasmids. We present an MG1655 ΔrecA single-locus knockout that avoids multimer production. These results can aid synthetic biologists in improving design and reliability of plasmid-based circuits.

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防止常用合成生物学质粒中多聚体的形成。

质粒是基础研究和生物技术应用的重要工具。为了优化基于质粒的电路，控制质粒的完整性是至关重要的，包括质粒多聚体的形成。多聚体是整个质粒在复制过程中由于二聚体分解失败而形成的串联重复序列。多聚体可以影响合成电路的行为，尤其是那些包含dna编辑酶的合成电路。然而，多定时器的发生通常不进行分析。本文研究了克隆型（JM109）和野生型（MG1655）大肠杆菌中常见的4种质粒主干中多聚体的发生情况。我们发现多聚体只在MG1655中出现，多聚体存在的质粒比例随着质粒拷贝数和培养传代的增加而增加。相反，将多聚体转化为JM109可以产生不含单线态质粒的菌株。我们提出了MG1655 ΔrecA单位点基因敲除，避免了多位点的产生。这些结果可以帮助合成生物学家改进基于质粒的电路的设计和可靠性。

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.