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Bacteriophage recombineering in the lytic state using the lambda red recombinases.

Fehér T, Karcagi I, Blattner FR, Pósfai G - Microb Biotechnol (2011)

Bottom Line: Bacteriophages, the historic model organisms facilitating the initiation of molecular biology, are still important candidates of numerous useful or promising biotechnological applications.With the help of BRED, we removed a copy of mobile element IS1, shown to be active, from the genome of P1vir, a coliphage frequently used in genome engineering procedures.Overall, P1virdeltaIS provides a genome engineering vehicle free of IS contamination, and BRED is likely to serve as a generally applicable tool for engineering bacteriophage genomes in a wide range of taxa.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary. fehert@brc.hu

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Related in: MedlinePlus

A. PCR screening, using primers P1D and P1test, of plaques obtained upon phage recombineering. Lanes 4 and 11 contain PCR products that indicate the presence of P1virΔIS within the plaques. B. PCR verification of the shortening of the IS‐deleted region in a pure P1virΔIS lysate using primers P1D and P1 BP. M: 1 kb DNA ladder (Fermentas).
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f3: A. PCR screening, using primers P1D and P1test, of plaques obtained upon phage recombineering. Lanes 4 and 11 contain PCR products that indicate the presence of P1virΔIS within the plaques. B. PCR verification of the shortening of the IS‐deleted region in a pure P1virΔIS lysate using primers P1D and P1 BP. M: 1 kb DNA ladder (Fermentas).

Mentions: P1virΔIS was constructed by deleting IS1P1vir, as described in Experimental procedures. Briefly, the genomic segment containing the deletion joint was assembled using overlapping PCR fragments. A mixture of this DNA fragment and the genomic DNA of P1vir was electroporated into a host expressing the λ‐red recombinases, allowing assembly of both wt and IS‐free P1 genomes. Host cells were plated in soft agar, and the emergence of plaques indicated successful assembly of phage genomes, despite the inevitable presence of double‐stranded breaks in the P1 chromosome (no plaques appeared when transforming λ‐red recombinase‐negative cells with P1 DNA). Plaques were picked, and the agarose plugs were digested with agarase to increase the number of released virions. Screening was done using a primer that spans the deletion (P1test), and a primer that anneals to the unaltered part of the phage genome (P1D) (Fig. 3A). The ratio of P1 DNA to the linear DNA fragment, the agarase treatment, as well as the incubation time after electroporation were all varied to optimize the BRED process (Table S1). In an optimal case, four out of 24 plaques tested positive. One mixed plaque, identified this way, was re‐plated on a bacterial lawn to obtain plaques that are either purely wt or purely recombinants. Four out of 15 plaques turned out to harbour recombinant P1virΔIS. Phage lysates generated from such pure recombinant plaques were confirmed to carry the deletion by PCR, using primers P1 BP and P1D (Fig. 3B). P1virΔIS was grown on MDS42+MD64 for another round of IS‐screening, performed using the White Glove IS Detection Kit. The test kit indicated no further copies of IS1, or any other transposable element of E. coli to be present in the phage genome (Fig. S1B).


Bacteriophage recombineering in the lytic state using the lambda red recombinases.

Fehér T, Karcagi I, Blattner FR, Pósfai G - Microb Biotechnol (2011)

A. PCR screening, using primers P1D and P1test, of plaques obtained upon phage recombineering. Lanes 4 and 11 contain PCR products that indicate the presence of P1virΔIS within the plaques. B. PCR verification of the shortening of the IS‐deleted region in a pure P1virΔIS lysate using primers P1D and P1 BP. M: 1 kb DNA ladder (Fermentas).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815324&req=5

f3: A. PCR screening, using primers P1D and P1test, of plaques obtained upon phage recombineering. Lanes 4 and 11 contain PCR products that indicate the presence of P1virΔIS within the plaques. B. PCR verification of the shortening of the IS‐deleted region in a pure P1virΔIS lysate using primers P1D and P1 BP. M: 1 kb DNA ladder (Fermentas).
Mentions: P1virΔIS was constructed by deleting IS1P1vir, as described in Experimental procedures. Briefly, the genomic segment containing the deletion joint was assembled using overlapping PCR fragments. A mixture of this DNA fragment and the genomic DNA of P1vir was electroporated into a host expressing the λ‐red recombinases, allowing assembly of both wt and IS‐free P1 genomes. Host cells were plated in soft agar, and the emergence of plaques indicated successful assembly of phage genomes, despite the inevitable presence of double‐stranded breaks in the P1 chromosome (no plaques appeared when transforming λ‐red recombinase‐negative cells with P1 DNA). Plaques were picked, and the agarose plugs were digested with agarase to increase the number of released virions. Screening was done using a primer that spans the deletion (P1test), and a primer that anneals to the unaltered part of the phage genome (P1D) (Fig. 3A). The ratio of P1 DNA to the linear DNA fragment, the agarase treatment, as well as the incubation time after electroporation were all varied to optimize the BRED process (Table S1). In an optimal case, four out of 24 plaques tested positive. One mixed plaque, identified this way, was re‐plated on a bacterial lawn to obtain plaques that are either purely wt or purely recombinants. Four out of 15 plaques turned out to harbour recombinant P1virΔIS. Phage lysates generated from such pure recombinant plaques were confirmed to carry the deletion by PCR, using primers P1 BP and P1D (Fig. 3B). P1virΔIS was grown on MDS42+MD64 for another round of IS‐screening, performed using the White Glove IS Detection Kit. The test kit indicated no further copies of IS1, or any other transposable element of E. coli to be present in the phage genome (Fig. S1B).

Bottom Line: Bacteriophages, the historic model organisms facilitating the initiation of molecular biology, are still important candidates of numerous useful or promising biotechnological applications.With the help of BRED, we removed a copy of mobile element IS1, shown to be active, from the genome of P1vir, a coliphage frequently used in genome engineering procedures.Overall, P1virdeltaIS provides a genome engineering vehicle free of IS contamination, and BRED is likely to serve as a generally applicable tool for engineering bacteriophage genomes in a wide range of taxa.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary. fehert@brc.hu

Show MeSH
Related in: MedlinePlus