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

  • Shawkat Ali
  • Guus Bakkeren
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Recombineering Binary vector Bacterial artificial chromosome Basidiomycete Ustilago Agrobacterium-mediated transformation 

Notes

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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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Pacific Agri-Food Research CentreAgriculture and Agri-Food CanadaSummerlandCanada
  2. 2.Department of BotanyUniversity of British ColumbiaVancouverCanada
  3. 3.Center for Desert Agriculture, Division of Biological and Environmental Sciences and EngineeringKing Abdullah University of Science and TechnologyThuwalSaudi Arabia

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