Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR.
Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR.
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DOI：
10.1186/s12934-016-0610-8
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发表时间：
2016-12-19
影响因子：
6.4
通讯作者：
Müller S
中科院分区：
文献类型：
--
作者：
Jahn M;Vorpahl C;Hübschmann T;Harms H;Müller S
质粒在实验室和工业环境中广泛用于分子克隆或蛋白质生产。不断的修饰产生了无数的质粒载体，而这些载体在平均拷贝数（PCN）和稳定性方面的特性却很少为人所知。决定PCN的关键因素是复制系统；目前使用的大多数复制系统属于少数不同的类，并且可以通过诸如标准欧洲矢量架构（Standard European Vector Architecture， SEVA）之类的存储库获得。在这项研究中，PCN是在一组7个基于seva的表达质粒中测定的，只是复制系统不同。所有构建体的平均PCN由微滴数字PCR测定，在宿主大肠杆菌中，每条染色体的PCN在2到40之间。此外，质粒编码的EGFP报告蛋白被用作评估单细胞水平上报告基因表达变异性的手段。只有一种质粒（RSF1010复制系统）的细胞表现出高度的异质性，EGFP强度呈明显的双峰分布，而其他质粒呈正态分布。对携带rsf1010的异种细胞群体和一个正态分布的群体（ColE1复制系统）进行分析，并根据EGFP强度对亚群体进行细胞分选。对于这两种质粒，低荧光亚群和高荧光亚群的PCN差异显著，ColE1和RSF1010的PCN分别在9.2 ~ 123.4和0.5 ~ 11.8之间。这里确定的一组标准化质粒的平均PCN通常处于先前报道范围的低端，与异质性的程度无关。异质种群和同质种群的进一步表征表明，亚种群的PCN存在相当大的差异。因此，我们提出了直接的分子证据，证明平均PCN并不代表单个细胞中质粒分子的真实数量。本文的在线版本（doi:10.1186/s12934-016-0610-8）包含补充材料，可供授权用户使用。
Plasmids are widely used for molecular cloning or production of proteins in laboratory and industrial settings. Constant modification has brought forth countless plasmid vectors whose characteristics in terms of average plasmid copy number (PCN) and stability are rarely known. The crucial factor determining the PCN is the replication system; most replication systems in use today belong to a small number of different classes and are available through repositories like the Standard European Vector Architecture (SEVA). In this study, the PCN was determined in a set of seven SEVA-based expression plasmids only differing in the replication system. The average PCN for all constructs was determined by Droplet Digital PCR and ranged between 2 and 40 per chromosome in the host organism Escherichia coli. Furthermore, a plasmid-encoded EGFP reporter protein served as a means to assess variability in reporter gene expression on the single cell level. Only cells with one type of plasmid (RSF1010 replication system) showed a high degree of heterogeneity with a clear bimodal distribution of EGFP intensity while the others showed a normal distribution. The heterogeneous RSF1010-carrying cell population and one normally distributed population (ColE1 replication system) were further analyzed by sorting cells of sub-populations selected according to EGFP intensity. For both plasmids, low and highly fluorescent sub-populations showed a remarkable difference in PCN, ranging from 9.2 to 123.4 for ColE1 and from 0.5 to 11.8 for RSF1010, respectively. The average PCN determined here for a set of standardized plasmids was generally at the lower end of previously reported ranges and not related to the degree of heterogeneity. Further characterization of a heterogeneous and a homogeneous population demonstrated considerable differences in the PCN of sub-populations. We therefore present direct molecular evidence that the average PCN does not represent the true number of plasmid molecules in individual cells. The online version of this article (doi:10.1186/s12934-016-0610-8) contains supplementary material, which is available to authorized users.
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4.3
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