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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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Updated: May 7, 2026

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Published on: November 12, 2012

Engineering large functional plasmids for biosafety.

Chris Cangelosi¹, Caroline Shank, Clayton Santiago

¹Department of Biology, Villanova University, 800 Lancaster Avenue, Villanova, PA 19085, USA.

|September 24, 2013

Summary

This summary is machine-generated.

Researchers developed a new recombineering method to efficiently remove unwanted genes from large bacterial plasmids. This method uses non-antibiotic selection, enhancing the safety and utility of engineered plasmids for medical and environmental applications.

Keywords:

FLP FRT R995 Recombineering asd trpE

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Last Updated: May 7, 2026

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

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Published on: November 12, 2012

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

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Published on: November 25, 2015

Area of Science:

Molecular Biology
Synthetic Biology
Genetic Engineering

Background:

Large bacterial plasmids engineered for specific functions often contain antibiotic resistance genes and conjugation systems.
Removal of these elements is crucial for biosafety in medical and environmental applications.
Existing methods for large plasmid manipulation are often inefficient or infeasible.

Purpose of the Study:

To develop a convenient in vivo recombineering strategy for the sequential removal of multiple genes from large bacterial plasmids.
To implement non-antibiotic selection methods for marker removal and plasmid maintenance.
To enhance the practical utility and biosafety of engineered large plasmids.

Main Methods:

Utilized lambda Red recombination with PCR products encoding trpE and asd genes for selection.
Employed FLP/FRT-mediated recombination for marker removal.
Applied the strategy to large plasmids (R995+SPI-1 and R995+SPI-2) encoding type III secretion systems.

Main Results:

Successfully removed antibiotic resistance markers and conjugation genes from large plasmids.
Demonstrated the first use of trpE gene/tryptophan prototrophy for recombineering selection.
Resulting plasmids were functional, stably maintained, and conjugation-defective.
Transferred plasmids via electroporation.

Conclusions:

Developed a novel and efficient recombineering approach for large plasmid engineering.
The method enhances biosafety by removing unwanted genes using non-antibiotic selection.
This strategy increases the stability and practical utility of engineered large plasmids for various applications.