Details zur Publikation

Referenztyp Zeitschriften
DOI / URL Link
Titel (primär) Accurate determination of plasmid copy number of flow-sorted cells using droplet digital PCR
Autor Jahn, M.; Vorpahl, C.; Türkowsky, D.; Lindmeyer, M.; Bühler, B.; Harms, H.; Müller, S.;
Journal / Serie Analytical Chemistry
Erscheinungsjahr 2014
Department UMB; METABOX;
Band/Volume 86
Heft 12
Sprache englisch;
POF III (gesamt) R31;
UFZ Querschnittsthemen RU4
Abstract

Many biotechnological processes rely on the expression of a plasmid-based target gene. A constant and sufficient number of plasmids per cell is desired for efficient protein production. To date, only a few methods for the determination of plasmid copy number (PCN) are available, and most of them average the PCN of total populations disregarding heterogeneous distributions. Here, we utilize the highly precise quantification of DNA molecules by droplet digital PCR (ddPCR) and combine it with cell sorting using flow cytometry. A duplex PCR assay was set up requiring only 1000 sorted cells for precise determination of PCN. The robustness of this method was proven by thorough optimization of cell sorting, cell disruption, and PCR conditions. When non plasmid-harboring cells of Pseudomonas putida KT2440 were spiked with different dilutions of the expression plasmid pA-EGFP_B, a PCN from 1 to 64 could be accurately detected. As a proof of principle, induced cultures of P. putida KT2440 producing an EGFP-fused model protein by means of the plasmid pA-EGFP_B were investigated by flow cytometry and showed two distinct subpopulations, fluorescent and nonfluorescent cells. These two subpopulations were sorted for PCN determination with ddPCR. A remarkably diverging plasmid distribution was found within the population, with nonfluorescent cells showing a much lower PCN (≤1) than fluorescent cells (PCN of up to 5) under standard conditions.

ID 15069
dauerhafte UFZ-Verlinkung http://www.ufz.de/index.php?en=20939&ufzPublicationIdentifier=15069
Jahn, M., Vorpahl, C., Türkowsky, D., Lindmeyer, M., Bühler, B., Harms, H., Müller, S. (2014):
Accurate determination of plasmid copy number of flow-sorted cells using droplet digital PCR
Anal. Chem. 86 (12), 5969 - 5976