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Related Concept Videos

Plasmids01:28

Plasmids

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Plasmids are extrachromosomal DNA molecules found in bacteria, archaea, and some eukaryotic microbes like yeast. These small, circular DNA structures typically contain fewer than 30 genes, although some may exist linearly. Plasmids vary in their number within a cell, known as copy number. Single-copy plasmids are present in one copy per cell and multi-copy plasmids are present in multiple copies, reaching over 100 copies per cell.Plasmids usually replicate independently of the chromosomal DNA...
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Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines
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Plasmid fermentation process for DNA immunization applications.

Aaron E Carnes1, James A Williams

  • 1Nature Technology Corporation, 4701 Innovation Drive, Lincoln, NE, 68521, USA, acarnes@natx.com.

Methods in Molecular Biology (Clifton, N.J.)
|April 10, 2014
PubMed
Summary
This summary is machine-generated.

This study presents a simple laboratory-scale fermentation process for producing high-quality plasmid DNA. This method is ideal for in-house production for animal studies, ensuring purity and yield.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Fermentation Technology

Background:

  • Plasmid DNA (pDNA) requires high purity and quality for immunization.
  • Upstream fermentation significantly impacts downstream purification efficiency.
  • Clinical facilities have validated pDNA fermentation processes, but lab-scale methods are needed for research.

Purpose of the Study:

  • To describe a simple, inexpensive laboratory-scale fed-batch fermentation process for producing bacterial cell paste rich in high-quality plasmid DNA.
  • To provide protocols for cell banking and seed culture preparation to optimize pDNA yield and quality.
  • To enable in-house production of research-grade pDNA for animal efficacy studies.

Main Methods:

  • Utilized a fed-batch fermentation strategy with constant feeding.
  • Achieved medium cell density with continuous plasmid amplification.
  • Detailed cell banking and seed culture preparation protocols.

Main Results:

  • Successfully produced bacterial cell paste enriched with high-quality plasmid DNA.
  • Demonstrated a constant feeding strategy leading to increased plasmid amplification.
  • Established protocols suitable for producing 100 mg to 1.5 g of research-grade pDNA from a 10 L fermentor.

Conclusions:

  • A simple laboratory-scale fermentation process can yield high-quality plasmid DNA.
  • Optimized upstream protocols are crucial for maximizing pDNA yield and purity.
  • This method supports cost-effective in-house pDNA production for preclinical research.