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New Method Combines Cell Sorting, ddPCR to Determine Plasmid Copy Number

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NEW YORK (GenomeWeb) — Plasmids are a simple system for producing recombinant proteins, but variability in the number of plasmid copies per cell limits expression. Now, researchers in Germany have developed a method to accurately ascertain plasmid copy number, or PCN, using flow cytometry and droplet digital PCR.

In a study published in Analytical Chemistry last month, a group at the Helmholtz Centre for Environmental Research in Leipzig and Technische Universität Dortmund was able to determine PCN in both a relative and absolute manner with higher precision than before. The study suggested precise plasmid determination has the potential to improve productivity in laboratory and commercial applications.

First author on the study Michael Jahn, a doctoral student in the department of environmental microbiology at Helmholtz, told PCR Insider in an email that many companies and researchers may use non-optimized strains of plasmids and disregard population heterogeneity, and therefore "will suffer from reduced yield."

Although plasmids were the expression vector du jour about 30 years ago, Jahn explained, they have been somewhat usurped by other techniques, such as genome-based expression or "from-scratch-design" of pathways and organisms.

"However, we think plasmids are still extremely interesting tools because they're easy to construct and provide high gene dosage, depending on the copy number," he added.

Jahn pointed to industrial uses of plasmids to manufacture pharmaceutical peptides and DNA for vaccines. "Especially for the latter, consistently high copy numbers are essential," Jahn said.

His own work is aimed at helping develop a bio-based economy, "where production of bulk and fine chemicals is done by microbes based on sustainable resources, in contrast to the petrol-based chemical industry," he said. "Right now, bio-based processes are getting more and more attention, but are often still not competitive. We are trying to analyze and optimize those bioprocesses, to accelerate this transition in the long run."

The new method of determining PCN is unique in combining and optimizing three existing methods.

The group first hammered out the sample prep. Their template was made up of as little as a few hundred whole Pseudomonas putida KT2440 cells spiked with different dilutions of the expression plasmid pA-EGFP_B. Using whole bacterial cells as starting material can create problems, but the group devised a way to reliably extract DNA, as well as reduce the impact of PCR inhibitors, involving simple heat treatment.

Cell sorting by flow cytometry separated EGFP-expressing from non-expressing cells. If a cell fluoresced, it contained plasmid. But some non-fluorescing cells may also have contained plasmid, and, in either case, how many copies of plasmid per cell was unknown. Previous studies have suggested heterogeneity in PCN, including simple bimodal distributions, according to the study.

A duplex ddPCR step allowed simultaneous quantification of bacterial genomic DNA and plasmid DNA in a small aliquot of cells at a time. Jahn said his group hopes to push this to single-cell analysis soon.

Overall, Jahn said the digital method was an improvement over traditional strategies. In ddPCR, "you have some extra steps compared to classic qRT-PCR," he said, noting that ddPCR requires disposable microfluidic chips and some additional liquid handling to create the water in oil droplet emulsion. "The data is not directly generated during the PCR run like in qRT-PCR, but you need to analyze the 96-well plate afterwards, taking up to 3 hours for a full plate," he added. However, "Apart from this extra work, we encountered much higher reproducibility compared to qRT-PCR and ... less spurious samples."

The group used the QX100 platform from Bio-Rad, which Jahn said they'd learned about at a conference and were thus "happy that our institute decided to obtain this system."

He said that they also noted rare "false-positive" results, as previously reported in PCR Insider. In runs of purified DNA, "We think these [control] droplets do not really contain DNA, because fluorescence is often in a different range than 'real' droplets. Most likely, these are dust particles," Jahn said. "However, our negative controls for cell sorting are sorted beads, which take the same way through the cytometer as real cells. These negative controls may indeed contain some real DNA fragments which are flushed from the cytometer's tubing."

Overall, the results of the study showed "a remarkably diverging plasmid distribution" with a PCN ranging between one and five per cell. Additionally, Jahn said that the PCN they discovered was also much lower than estimated in the classical papers on that particular plasmid family. "It is very well possible that many copy numbers floating around in literature are overestimated, due to the different techniques used."

The group plans to use this workflow in their lab from now on and possibly adapt it for RNA detection, Jahn said. They'd also like to reduce the input sample cell numbers, potentially to enable PCN determination on a single-cell basis.

While they have no commercial intentions for this method as yet, they are "willing and interested in collaborations with other researchers or companies who [would] like to know a bit more about the expression systems they use," said Jahn.

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