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Chapter 34 : Genetic Manipulations Using Phages

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Genetic Manipulations Using Phages, Page 1 of 2

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Abstract:

Lytic (or virulent) phages usually have only a single outcome upon infection of a bacterial host: reproduction of the virus and lysis of the bacterium. As a consequence, plaques appear as perfectly clear areas of infection, within which all of the bacterial cells are dead. The role of phage genetics in microbiology has expanded with the realization that we understand only a small fraction of bacterial diversity. Microbes such as and have been deeply studied and to great effect, but as our attention has turned to the broader bacterial community, new bacteria have brought new genetic challenges. This chapter describes some common phage methods and approaches that can be applied to virtually any bacterial host. Several collections of mycobacteriophages had been assembled previously with a view to using their host-range specificities for the typing of clinical isolates. One of these phages had been shown to mediate generalized transduction in , and researchers have achieved uptake of phage DNA by . The chapter also describes how phages can be isolated, purified, and genomically characterized, how shuttle phasmids can be constructed and utilized, and how phages can be used to construct a variety of genetic tools. The potential for phage genetics in any bacterial host is vast, and the approaches and methods described in the chapter represent just a small part of what bacteriophages have to offer to the microbial genetic engineer.

Citation: Hatfull G, Jacobs-Sera D, Larsen M, Jacobs W. 2007. Genetic Manipulations Using Phages, p 825-838. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch34

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Figures

Image of FIGURE 1
FIGURE 1

Mycobacteriophage plaque morphologies on lawns. (A) Small clear plaques (Mycobacteriophage Oasis). (B) Large clear plaques (Mycobacteriophage Hammer). (C) Turbid plaques (Mycobacteriophage RedRock).

Citation: Hatfull G, Jacobs-Sera D, Larsen M, Jacobs W. 2007. Genetic Manipulations Using Phages, p 825-838. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch34
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Image of FIGURE 2
FIGURE 2

Construction of shuttle phasmids. Shuttle phasmids are chimeric molecules that replicate as extrachromosomal plasmids in and as phages in another bacterial host. Construction details are provided in the text.

Citation: Hatfull G, Jacobs-Sera D, Larsen M, Jacobs W. 2007. Genetic Manipulations Using Phages, p 825-838. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch34
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Image of FIGURE 3
FIGURE 3

Specialized transduction. Regions flanking the upstream and downstream regions of “Your favorite gene” () are cloned into a hygromycin-containing cassette on a plasmid containing both lambda sites and mycobacteriophage sites. is transfected at 30°C to allow for production of a high-titer phage lysate containing the Δ:: allele. The high-titer phage lysate is then used to transduce at 37°C (nonpermissive temperature for phage lysis) to deliver Δ:: substrate to the cell for subsequent allelic exchange.

Citation: Hatfull G, Jacobs-Sera D, Larsen M, Jacobs W. 2007. Genetic Manipulations Using Phages, p 825-838. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch34
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