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    Rapid Targeted Gene Disruption in Bacillus Anthracis

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    2013_09_Saldanha.pdf (785.5Kb)
    Date
    2013-09
    Author
    Saldanha, Roland J.
    Pemberton, Adin
    Shiflett, Patrick
    Perutka, Jiri
    Whitt, Jacob T.
    Ellington, Andrew
    Lambowitz, Alan M.
    Kramer, Ryan
    Taylor, Deborah
    Lamkin, Thomas J.
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    Abstract
    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.
    Department
    Cellular and Molecular Biology
    Subject
    group-ii intron
    escherichia-coli
    genome sequence
    dna
    endonuclease
    bacteria
    mobility
    protein
    rna
    transcription
    biotechnology & applied microbiology
    URI
    http://hdl.handle.net/2152/43367
    Citation
    Saldanha, Roland J., Adin Pemberton, Patrick Shiflett, Jiri Perutka, Jacob T. Whitt, Andrew Ellington, Alan M. Lambowitz, Ryan Kramer, Deborah Taylor, and Thomas J. Lamkin. "Rapid targeted gene disruption in Bacillus anthracis." BMC biotechnology, Vol. 13, No. 1 (Sep., 2013): 72.
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