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Updated: Aug 19, 2026
CRISPR-based Shuttle Cloning: A High-throughput Cloning Method
Published on: June 13, 2025
Versatile Rhodococcus equi-Escherichia coli shuttle vectors
Michael W Mangan1, Gavin A Byrne, Wim G Meijer
1Department of Industrial Microbiology, Conway Institute for Biomolecular and Biomedical Reseach, University College Dublin, Dublin 4, Ireland.
Abstract:
Rhodococcus equi is an intracellular pathogen of macrophages, causing disease in young foals, humans, and sporadically other animals. Although R. equi is easy to grow and manipulate, the analysis of virulence is hampered by a lack of molecular tools. This paper describes the development of a number of versatile plasmids for use in R. equi. Plasmids pREV2 and pREV5 use origins of replication derived from the Mycobacterium fortuitum plasmids pAL5000 and pMF1. These plasmids and their derivatives are compatible in R. equi, allowing their use for analysis of gene function in trans. The stability of these plasmids in R. equi in the absence of selection for the plasmid borne antibiotic resistance markers, and their integrity following passage through Escherichia coli and R. equi was determined.
Insights
Researchers developed new plasmids for Rhodococcus equi, an important animal and human pathogen. These molecular tools will aid in understanding R. equi virulence and developing control strategies.
Area of Science:
- Microbiology
- Molecular Biology
- Veterinary Medicine
Background:
- Rhodococcus equi is an intracellular pathogen affecting foals, humans, and other animals.
- Understanding R. equi virulence is crucial but limited by a lack of molecular tools.
- Development of versatile plasmids is needed for genetic manipulation of R. equi.
Purpose of the Study:
- To develop and characterize versatile plasmids for use in Rhodococcus equi.
- To enable the analysis of gene function in trans within R. equi.
- To assess plasmid stability and integrity in R. equi and Escherichia coli.
Main Methods:
- Construction of plasmids (pREV2, pREV5) using origins of replication from Mycobacterium fortuitum plasmids.
- Testing plasmid compatibility in R. equi.
- Evaluating plasmid stability in R. equi without antibiotic selection.
- Assessing plasmid integrity after passage through E. coli and R. equi.
Main Results:
- Development of compatible plasmids (pREV2, pREV5) for R. equi.
- Demonstration of plasmid utility for trans-acting gene function analysis.
- Assessment of plasmid stability and integrity under various conditions.
Conclusions:
- The developed plasmids provide essential molecular tools for R. equi research.
- These plasmids facilitate the study of R. equi virulence mechanisms.
- The tools will advance understanding and control of R. equi infections.
