Abstract
BackgroundPlasmids 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).ResultsIn 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0610-8) contains supplementary material, which is available to authorized users.
Highlights
Plasmids are widely used for molecular cloning or production of proteins in laboratory and industrial settings
Cloning of p2X4‐a styrene monooxygenase (styA)-EGFP styB (AEB) vector series We used the Standard European Vector Architecture (SEVA) platform to create a range of new plasmid vectors being completely identical except for the replication system [11]
Of the nine replication systems in the SEVA repository, we omitted the first one, R6K, as it is intended for suicide vectors, and the last one, pBR322
Summary
Plasmids are widely used for molecular cloning or production of proteins in laboratory and industrial settings. For biotechnological applications it is highly desirable to know the range of copy numbers that can be expected from a particular vector, as the gene dosage can be crucial for efficient protein production [3]. It must be noted that this behavior is different in lowcopy plasmids that carry active partitioning systems to ensure faithful distribution of one or few plasmid copies from mother to daughter cells. This reduces heterogeneity, no such system was included in this study. As plasmids follow a discrete distribution, a low mean PCN may lead to higher cell to cell heterogeneity regarding PCN and gene expression, as e.g. shown by Kittleson and colleagues using a library of 20 rep mutants [8]
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