Vaisbourd Elizabeth, Bren Anat, Alon Uri, Glass David S
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel 76100.
ACS Synth Biol. 2025 Apr 18;14(4):1309-1315. doi: 10.1021/acssynbio.4c00508. Epub 2025 Mar 18.
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 . 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 single-locus knockout that avoids multimer production. These results can aid synthetic biologists in improving design and reliability of plasmid-based circuits.
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，控制质粒完整性至关重要，这包括质粒多聚体的形成。多聚体是在复制过程中由于二聚体解析失败而形成的整个质粒的串联重复。多聚体可影响合成电路的行为，尤其是那些包含DNA编辑酶的电路。然而，多聚体的出现并不常被检测。在这里，我们调查了四种常用的质粒骨架在克隆菌株（JM109）和野生型菌株（MG1655）中多聚体的出现情况。我们发现多聚体仅在MG1655中显著出现，作为多聚体存在的质粒比例随着质粒拷贝数和培养传代次数的增加而增加。相比之下，将多聚体转化到JM109中可产生不含单体质粒的菌株。我们提出了一种避免多聚体产生的MG1655单基因敲除方法。这些结果有助于合成生物学家提高基于质粒的电路的设计和可靠性。
