title: Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR.
abstract: BACKGROUND
  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).
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Helmholtz-Centre for Environmental Research-UFZ, Permoserstraße 15, 04318, Leipzig, Germany.|School of Biotechnology, Science for Life Laboratory, KTH-Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Michael
authors.identifier: http://orcid.org/0000-0002-3913-153X
authors.initials: M
authors.lastname: Jahn
authors.affiliation: Helmholtz-Centre for Environmental Research-UFZ, Permoserstraße 15, 04318, Leipzig, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Vorpahl
authors.affiliation: Helmholtz-Centre for Environmental Research-UFZ, Permoserstraße 15, 04318, Leipzig, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hübschmann
authors.affiliation: Helmholtz-Centre for Environmental Research-UFZ, Permoserstraße 15, 04318, Leipzig, Germany.
authors.forename: Hauke
authors.initials: H
authors.lastname: Harms
authors.affiliation: Helmholtz-Centre for Environmental Research-UFZ, Permoserstraße 15, 04318, Leipzig, Germany. susann.mueller@ufz.de.
authors.forename: Susann
authors.initials: S
authors.lastname: Müller
mesh_terms: D056915:DNA Copy Number Variations; D004926:Escherichia coli / Q000821:virology; D005434:Flow Cytometry / Q000379:methods*; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*
keywords: Cell sorting; EGFP; Escherichia coli; Origin of replication; Plasmid copy number; Population heterogeneity; Replication system; SEVA; Sub-population; Variability