Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR

Abstract

Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a straightforward and reliable method to quantify the plasmid copy number. Therefore we believe that the ddPCR designed in this study will be widely used for any plasmid copy number calculation in the future.

A Critical Region in the FlaA Flagellin Facilitates Filament Formation of the Vibrio cholerae Flagellum

“…(pBR322 ori; ϳ20 copies per cell [27]) in the ΔflaA-E strain in the presence of 0.2% arabinose. Motility increased with increasing copy number of flaA expressed from flaAp, demonstrating that relatively large amounts of FlaA are necessary for monoflagellin filament formation.…”

RepB C-terminus mutation of a pRi-repABC binary vector affects plasmid copy number in Agrobacterium and transgene copy number in plants

“…The difference in plasmid copy number between Oltmanns et al [ 21 ] and this study may result from the assay method, Agrobacterium strain, or growth time difference. Plasmid copy numbers are determined primarily by their replication origins, which show significant cell-to-cell variation within every population [ 32 ]. Oltmanns et al [ 21 ] reported 15–20 copies/cell with pTF::Ri carrying RepB Y299H and repB T486C mutations in A .…”

RepB C-terminus mutation of an ori pRi vector affects plasmid copy number inAgrobacteriumand transgene copy number in plants

“…The difference in plasmid copy number between Oltmanns et al [14] and this study may result from the assay method, Agrobacterium strain, or growth time difference. Plasmid copy numbers are determined primarily by their replication origins, which show significant cell-to-cell variation within every population [28]. Oltmanns et al [14] reported 15-20 copies/cell with pTF::Ri carrying RepB Y299H and repB T486C mutations in A. tumefaciens EHA105 using a DNA blot assay, whereas in this study we estimated that both the single and the double mutations in a nopaline-type ABI strain have 9-10 copies/cell, using a ddPCR assay.…”