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Conversion of BAC Clones into Binary BAC (BIBAC) Vectors and Their Delivery into Basidiomycete Fungal Cells Using Agrobacterium tumefaciens

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Bacterial Artificial Chromosomes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1227))

Abstract

The genetic transformation of certain organisms, required for gene function analysis or complementation, is often not very efficient, especially when dealing with large gene constructs or genomic fragments. We have adapted the natural DNA transfer mechanism from the soil pathogenic bacterium Agrobacterium tumefaciens, to deliver intact large DNA constructs to basidiomycete fungi of the genus Ustilago where they stably integrated into their genome. To this end, Bacterial Artificial Chromosome (BAC) clones containing large fungal genomic DNA fragments were converted via a Lambda phage-based recombineering step to Agrobacterium transfer-competent binary vectors (BIBACs) with a Ustilago-specific selection marker. The fungal genomic DNA fragment was subsequently successfully delivered as T-DNA through Agrobacterium-mediated transformation into Ustilago species where an intact copy stably integrated into the genome. By modifying the recombineering vector, this method can theoretically be adapted for many different fungi.

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Acknowledgment

We thank Dr. Frank Takken, University of Amsterdam, for the plasmid pFT41 and pioneering work [16] and Dr. Neal Copeland, National Cancer Institute, Frederick, MD, for E. coli strain SW102 [4]. This work was supported by a Natural Sciences and Engineering Research Council of Canada grant to G. Bakkeren.

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Authors and Affiliations

  1. Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, 5000, Summerland, BC, Canada, V0H 1Z0

    Shawkat Ali & Guus Bakkeren Ph.D.

  2. Department of Botany, University of British Columbia, Vancouver, BC, Canada, V6T 1Z4

    Shawkat Ali

  3. Center for Desert Agriculture, Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal, 23955-6900, Saudi Arabia

    Shawkat Ali

Authors
  1. Shawkat Ali
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  2. Guus Bakkeren Ph.D.
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Correspondence to Guus Bakkeren Ph.D. .

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Editors and Affiliations

  1. Monash University Malaysia, Bandar Sunway, Malaysia

    Kumaran Narayanan

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Ali, S., Bakkeren, G. (2015). Conversion of BAC Clones into Binary BAC (BIBAC) Vectors and Their Delivery into Basidiomycete Fungal Cells Using Agrobacterium tumefaciens . In: Narayanan, K. (eds) Bacterial Artificial Chromosomes. Methods in Molecular Biology, vol 1227. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1652-8_9

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  • DOI: https://doi.org/10.1007/978-1-4939-1652-8_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1651-1

  • Online ISBN: 978-1-4939-1652-8

  • eBook Packages: Springer Protocols

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