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Construction of a high-efficiency shuttle vector for Histophilus somni
Indra Sandal1, Mohamed N Seleem, Shaadi F Elswaifi
1Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, United States.
Journal of Microbiological Methods
|May 27, 2008
Summary
Researchers developed a new plasmid, pNS3K, to improve genetic manipulation of Histophilus somni. This enhanced vector transforms bacteria 100-fold more efficiently, aiding in understanding and modifying this important pathogen.
Area of Science:
- Bacteriology
- Molecular Biology
- Genetics
Background:
- Histophilus somni presents challenges for genetic manipulation due to its robust restriction-modification system.
- Existing vectors, like the broad host-range shuttle plasmid pLS88, exhibit limited transformation efficiency in H. somni.
Purpose of the Study:
- To engineer an improved shuttle vector for enhanced genetic manipulation of Histophilus somni.
- To increase the transformation efficiency of H. somni compared to previously available vectors.
Main Methods:
- Construction of an improved pLS88 derivative, designated pNS3K.
- Transformation efficiency assays of pNS3K in H. somni strain 2336.
- Cloning and expression of the lipooligosaccharide biosynthesis gene lob-2A using pNS3K.
Main Results:
- The novel pNS3K vector demonstrated a 100-fold increase in transformation efficiency in H. somni strain 2336 compared to pLS88.
- Further enhancement of transformation efficiency was observed when pNS3K was isolated from H. somni and retransformed.
- Successful cloning and expression of the lob-2A gene in H. somni strain 129Pt, demonstrating vector functionality.
Conclusions:
- The pNS3K shuttle vector significantly enhances the genetic manipulation capabilities of Histophilus somni.
- pNS3K represents a valuable tool for future research involving genetic modification of H. somni, including functional gene studies.
