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Chapter 44 : Temperate Phage Vectors

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Abstract:

Temperate phages have been widely used as tools for genetic manipulation and analysis of . There are two very different approaches to cloning in temperate phage vectors in . Direct transfection is similar to the familiar shotgun cloning methods used in . Cloning the gene of interest depends on being able to select or detect a recombinant carrying the desired gene. This approach has been very successful, particularly for cloning sporulation genes. Prophage transformation is an ingenious cloning system that is unique to and takes advantage of the efficient transformation of this organism with linear DNA fragments. Vectors in which the products of a cloned gene are expressed are useful for two purposes. First, they can be used to identify the product of the cloned gene. It was discovered that the cloning site used in most of that the successful ø105 transfection vectors actually lies within a phage transcription unit. The second application for phage expression vectors lies in the overexpression of cloned genes for purposes of protein purification. There are a number of interesting phages in but that progress in understanding their biology lags far behind our understanding of the equivalent phages of .

Citation: Errington J. 1993. Temperate Phage Vectors, p 645-650. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch44

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Bacillus subtilis
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Escherichia coli
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Bacillus subtilis
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Escherichia coli
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Bacillus subtilis
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Escherichia coli
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Figures

Image of Figure 1
Figure 1

Schematic drawing of the structural and functional organization of phage cloning vector 105J106. The vegetative phage genome is represented by the central bar, with distances (in kilobase pairs) from its left end noted below the bar. Immediately above the bar are the positions of RI restriction sites. The fragments generated are labeled with standard one-letter designations (see reference for a detailed restriction map). The modifications of wild-type $105 incorporated into vector 105J106 are shown at the top. The polylinker inserted into a nonessential site in the phage contains unique restriction sites for (?), (X), and (S), the last having been made unique by removal of a site to the right of the genome (denoted [Sa/I]). A deletion removing 4 kbp of nonessential phage DNA (Δ 4kb) allows for the insertion of exogenous DNA fragments without exceeding the packaging limits of the phage. Approximate positions of regions of the phage genome devoted to particular functions are indicated below the bar. denotes the site at which the phage DNA integrates into the host chromosome by site-specific recombination ( ). The vegetative genome has single-stranded complementary termini that allow circularization of the genome prior to integration into the host chromosome ( ).

Citation: Errington J. 1993. Temperate Phage Vectors, p 645-650. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch44
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Image of Figure 2
Figure 2

Schematic illustration of the principles underlying the prophage transformation method ( ). Fragments of phage DNA and the target DNA to be cloned are ligated at a high DNA concentration and an approximately 1:1 molar ratio to form concatemeric molecules. Some of the molecules will have the structure shown at the top. Recombination between the fragments of phage DNA and their homologous sequences in the host prophage leads to insertion of target DNA in place of a segment of prophage DNA. Recombinants with the appropriate genotype can either be selected directly, as in this instance, or be generated at random if a selectable marker is placed at the inner margin of one of the fragments of phage DNA. In the original procedure, phage DNA fragments were derived by digestion of total phage DNA, sometimes generating many fragments, only a proportion of which would generate viable recombinant prophages. It is now more usual to provide specific fragments of cloned phage DNA flanking dispensable regions of the prophage genome.

Citation: Errington J. 1993. Temperate Phage Vectors, p 645-650. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch44
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Image of Figure 3
Figure 3

Schematic illustration of an expression system based on the defective prophage PBSX. The circular plasmid at the top of the diagram can be grown and manipulated in but cannot replicate autonomously in Transformants of selected on the basis of the plasmid-borne resistance marker arise by Campbell-type integration of the plasmid into the PBSX prophage via its region of homology (indicated by the black box). The prophage carries the mutation, which allows thermoinduction of the strong phage promoter. After plasmid integration, this promoter drives expression of the cloned gene. In addition, a transcription terminator in the plasmid blocks transcription of downstream phage genes, including genes required for host cell lysis.

Citation: Errington J. 1993. Temperate Phage Vectors, p 645-650. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch44
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Image of Figure 4
Figure 4

Schematic illustration of an expression system based on phage 105 ( ). Phage 105MU209 has an insertion consisting of a reporter gene followed by a selectable chloramphenicol resistance gene. The structure of the insertion is complex and not yet fully resolved. However, lies downstream from a strong phage promoter that can be induced about 100-fold by thermoinduction of the prophage, which carries the mutation. The insertion also blocks the host cell lysis that normally follows phage induction. To express other proteins in this system, the gene and part of the chloramphenicol resistance gene are first replaced with an erythromycin resistance gene (A). The latter is then replaced by the gene to be expressed (in this case, –lactamase I [β–I] from B. cereus) by selection for the full-length chloramphenicol resistance gene (B).

Citation: Errington J. 1993. Temperate Phage Vectors, p 645-650. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch44
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References

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