Rapid targeted gene disruption in Bacillus anthracis
| Autor: | Patrick Shiflett, Adin Pemberton, Ryan M. Kramer, Deborah Taylor, Jiri Perutka, Jacob T Whitt, Alan M. Lambowitz, Roland Saldanha, Thomas J. Lamkin, Andrew D. Ellington |
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| Rok vydání: | 2013 |
| Předmět: |
DNA
Bacterial Genetic Vectors Biology Genome 03 medical and health sciences Plasmid Escherichia coli Cloning Molecular Selection Genetic Gene Pathogen 030304 developmental biology Whole genome sequencing Genetics 0303 health sciences 030306 microbiology Methodology Article Gene targeting Chromosomes Bacterial biology.organism_classification Introns Bacillus anthracis Mutagenesis Insertional Susceptible individual Genes Bacterial Gene Targeting Nucleic Acid Conformation Biotechnology Plasmids |
| Zdroj: | BMC Biotechnology |
| ISSN: | 1472-6750 |
| Popis: | Background Anthrax is a zoonotic disease recognized to affect herbivores since Biblical times and has the widest range of susceptible host species of any known pathogen. The ease with which the bacterium can be weaponized and its recent deliberate use as an agent of terror, have highlighted the importance of gaining a deeper understanding and effective countermeasures for this important pathogen. High quality sequence data has opened the possibility of systematic dissection of how genes distributed on both the bacterial chromosome and associated plasmids have made it such a successful pathogen. However, low transformation efficiency and relatively few genetic tools for chromosomal manipulation have hampered full interrogation of its genome. Results Group II introns have been developed into an efficient tool for site-specific gene inactivation in several organisms. We have adapted group II intron targeting technology for application in Bacillus anthracis and generated vectors that permit gene inactivation through group II intron insertion. The vectors developed permit screening for the desired insertion through PCR or direct selection of intron insertions using a selection scheme that activates a kanamycin resistance marker upon successful intron insertion. Conclusions The design and vector construction described here provides a useful tool for high throughput experimental interrogation of the Bacillus anthracis genome and will benefit efforts to develop improved vaccines and therapeutics. |
| Databáze: | OpenAIRE |
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