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Use of Cosmid Libraries in Plant Transformations

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Agrobacterium Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 44))

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Abstract

Cosmid libraries are important tools for molecular analysis of plant genes because of their useful properties. Cosmids carry the cos site from the bacteriophage lambda (1–4); therefore, cosmid libraries have the advantage of being able to be packaged in vitro using commercially available highly efficient packaging extracts, just as lambda libraries can. The high efficiency of in vitro lambda packaging allows a cosmid library with large inserts to be more easily constructed than a simple plasmid library, which relies on transformation of competent Escherichia coli cells. In addition, cosmids contain an origin of replication for propagation in E. coli and possibly other bacteria. Since cosmids are propagated as plasmids, not as lambda phages, the genes for lambda reproduction are not needed; therefore, a cosmid vector can be much smaller than a lambda vector (Fig. 1). Since lambda phage packages about 37–53 kb of DNA (5), cosmids can have relatively large inserts of more than 40 kb, depending on vector sizes. Another advantage of cosmids is the ease with which to prepare the DNA because plasmid DNA is much more readily isolated than lambda phage DNA. Finally, the fact that cosmids are plasmids allows the recombinant molecules to be transferred from one bacterium to another through conjugation and from Agrobacterium to plant cells, as long as the necessary cis elements are present. Therefore, cosmid libraries offer a unique combination of very useful features, which make them important for many different molecular studies, including those of plant genes.

Comparison of lambda and cosmid clones. The vector for a lambda clone contains all of the genes required for the lytic life cycle of the lambda phage, including genes for phage DNA replication, for the phage structural proteins, and for lysis of E. coli cells. These genes are distributed in two regions, called left arm and right arm, usually about 20 and 9 kb in size, respectively. The lambda phage can package 37–53 kb; therefore, the insert sizes range from 8 kb (solid line) to 24 kb (solid and dashed lines). In contrast, cosmid clones propagate as plasmids, so the vector needs only to have a selectable marker (M) and an origin of replication (ori). So a cosmid vector can easily be less than 8 kb, allowing the inserts to be as long as 29 kb (solid line) to 45 kb (solid and dashed lines). Generally, a plasmid vector with a 400-bp fragment containing the cos site is a cosmid vector.

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Authors and Affiliations

  1. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Hong Ma

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  1. Hong Ma
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Editors and Affiliations

  1. Department of Molecular and Life Sciences, University of Abertay Dundee, Scotland, UK

    Kevan M. A. Gartland

  2. Department of Life Science, Plant Genetic Manipulation Group, University of Nottingham, UK

    Michael R. Davey

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© 1995 Humana Press Inc., Totowa, NJ

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Ma, H. (1995). Use of Cosmid Libraries in Plant Transformations. In: Gartland, K.M.A., Davey, M.R. (eds) Agrobacterium Protocols. Methods in Molecular Biology™, vol 44. Springer, Totowa, NJ. https://doi.org/10.1385/0-89603-302-3:351

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  • DOI: https://doi.org/10.1385/0-89603-302-3:351

  • Publisher Name: Springer, Totowa, NJ

  • Print ISBN: 978-0-89603-302-3

  • Online ISBN: 978-1-59259-531-0

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