Details zur Publikation
|Titel (primär)||Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR|
|Autor||Jahn, M.; Vorpahl, C.; Hübschmann, T.; Harms, H.; Müller, S.|
|Journal / Serie||Microbial Cell Factories|
|Seite von||art. 211|
|Keywords||Escherichia coli; SEVA; Replication system; Origin of replication; Plasmid copy number; EGFP; Population heterogeneity; Variability; Cell sorting; Sub-population|
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 single cell variability in reporter gene expression. Only one strain showed a high degree of heterogeneity (RSF1010 replication system) with a clear bimodal distribution of EGFP intensity while the others showed a normal distribution. The heterogeneous RSF1010-carrying strain and one normally distributed strain (ColE1 replication system) were further analyzed by sorting cells of sub-populations selected according to EGFP intensity. For both strains, 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 subpopulations. We therefore present direct molecular evidence that the average PCN does not represent the true number of plasmid molecules in individual cells.
|Jahn, M., Vorpahl, C., Hübschmann, T., Harms, H., Müller, S. (2016):
Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR
Microb. Cell. Fact. 15 , art. 211