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Plasmids

Plasmids

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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Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Published on: February 5, 2021

An improved method for including upper size range plasmids in metamobilomes.

Anders Norman¹, Leise Riber², Wenting Luo²

¹Section of Microbiology, Department of Biology, University of Copenhagen, Copenhagen, Denmark; Department of Earth and Planetary Science, University of California, Berkeley, California, United States of America.

|August 14, 2014

Summary

Multiple displacement amplification (MDA) biases plasmid DNA recovery, underrepresenting larger plasmids. A new electroelution method improves the recovery of medium-to-large plasmids for better ecological insights.

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Last Updated: Apr 25, 2026

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Published on: March 16, 2012

Area of Science:

Microbiology
Molecular Biology
Genomics

Background:

Plasmid DNA recovery is crucial for culture-independent studies of plasmid ecology.
Existing methods using multiple displacement amplification (MDA) show bias towards smaller DNA elements.
This bias leads to underrepresentation of larger plasmids in sequencing data.

Purpose of the Study:

To investigate the size bias of multiple displacement amplification (MDA) in plasmid DNA recovery.
To develop and validate a modified protocol for improved recovery of larger plasmids.
To enhance the understanding of plasmid ecology by enabling access to a wider range of plasmid sizes.

Main Methods:

Comparative analysis of plasmid DNA recovery using standard and modified protocols.
Utilized model plasmids (4.4 Kbp pBR322 and 56 Kbp pKJK10) to assess size-dependent amplification bias.
Incorporated an electroelution step prior to MDA in the modified protocol.
Validated the modified protocol using wastewater metagenomic data (metamobilomes).

Main Results:

Standard MDA significantly over-amplified small plasmids (pBR322) by 34-fold compared to large plasmids (pKJK10).
The modified protocol with electroelution enabled complete recovery of large plasmids (pKJK10).
Wastewater metamobilome analysis showed improved recovery of plasmids up to 30 Kbp median size with the modified protocol.
Gene functions associated with larger plasmids, like conjugation, were exclusively recovered using the modified method.

Conclusions:

Multiple displacement amplification (MDA) introduces significant size bias, favoring smaller plasmids.
An electroelution step effectively mitigates MDA's size bias, improving the recovery of medium-to-large plasmids.
The modified protocol enhances the comprehensive analysis of plasmid populations, providing better access to accessory genetic elements and improving plasmid ecology studies.