Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR.

Tadeusz Kaczorowski,Magdalena Plotka,Hideyuki Doi,Mateusz Wozniak

doi:10.1371/journal.pone.0169846

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.

Highlights

Plasmids play an important role in molecular biology and biotechnology, primarily as vectors for molecular cloning to facilitate the overproduction of recombinant proteins [1], and as PLOS ONE | DOI:10.1371/journal.pone.0169846 January 13, 2017Quantification of Plasmid Copy Number sophisticated nanotools for specialized applications in the genome engineering [2]
The DNA templates were isolated either with the QIAamp DNA mini kit or by the bead-beating method and diluted as presented on S1 Fig. Primer pairs used for bla and dxs amplification are listed in Table 1
The concentrations of bla and dxs of total DNA isolated either with the QIAamp DNA mini kit or by the bead-beating method are shown on Fig 1A and 1B, respectively

Summary

Introduction

Plasmids play an important role in molecular biology and biotechnology, primarily as vectors for molecular cloning to facilitate the overproduction of recombinant proteins [1], and as PLOS ONE | DOI:10.1371/journal.pone.0169846 January 13, 2017Quantification of Plasmid Copy Number sophisticated nanotools for specialized applications in the genome engineering [2]. Plasmid-oriented studies provide insights to improve understanding of DNA replication, maintenance and transfer strategies which are essential to all microorganisms [6, 7]. In this respect, among many features that characterize these mobile genetic elements, the one which defines the number of plasmid units that are contained inside one bacterial cell is especially important, both from a practical and a biological point of view. Quantification of the plasmid copy number is crucial in describing an expression system and exerts strong impact on protein production [8]. High-copy plasmids are preferred for efficient overproduction of recombinant proteins that do not affect the host viability, but in case of toxic or unstable proteins, usually low-copy plasmids are used [1]

Methods

Results

Discussion

Conclusion