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Whole-genome synthesis and characterization of viable S13-like bacteriophages.

Liu Y, Han Y, Huang W, Duan Y, Mou L, Jiang Z, Fa P, Xie J, Diao R, Chen Y, Ye Y, Yang R, Chen J, Sun X, Li Z, Tang A, Gui Y, Cai Z - PLoS ONE (2012)

Bottom Line: The results of sequencing showed the accuracy of these synthetic genomes.While no phenotypic differences among the variant strains were observed when grown in LB medium with CaCl(2), the S13/G4 chimera was found to be much more sensitive to the absence of calcium and to have a lower adsorption rate under calcium free condition.These results support an evolutional hypothesis which has been proposed that a homologous recombination event involving gene F of quite divergent ancestral lineages should be included in the history of the microvirid family.

View Article: PubMed Central - PubMed

Affiliation: Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Institute of Urology, Peking University Shenzhen Hospital, Shenzhen PKU-HKUST Medical Center, Shenzhen, China.

ABSTRACT

Background: Unprecedented progresses in high-throughput DNA sequencing and de novo gene synthesis technologies have allowed us to create living organisms in the absence of natural template.

Methodology/principal findings: The sequence of wild-type S13 phage genome was downloaded from GenBank. Two synonymous mutations were introduced into wt-S13 genome to generate m1-S13 genome. Another mutant, m2-S13 genome, was obtained by engineering two nonsynonymous mutations in the capsid protein coding region of wt-S13 genome. A chimeric phage genome was designed by replacing the F capsid protein open reading frame (ORF) from phage S13 with the F capsid protein ORF from phage G4. The whole genomes of all four phages were assembled from a series of chemically synthesized short overlapping oligonucleotides. The linear synthesized genomes were circularized and electroporated into E.coli C, the standard laboratory host of S13 phage. All four phages were recovered and plaques were visualized. The results of sequencing showed the accuracy of these synthetic genomes. The synthetic phages were capable of lysing their bacterial host and tolerating general environmental conditions. While no phenotypic differences among the variant strains were observed when grown in LB medium with CaCl(2), the S13/G4 chimera was found to be much more sensitive to the absence of calcium and to have a lower adsorption rate under calcium free condition.

Conclusions/significance: The bacteriophage S13 and its variants can be chemically synthesized. The major capsid gene of phage G4 is functional in the phage S13 life cycle. These results support an evolutional hypothesis which has been proposed that a homologous recombination event involving gene F of quite divergent ancestral lineages should be included in the history of the microvirid family.

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Related in: MedlinePlus

Images of the plaques (8 h, 37°C, 10 cm Petri dish).A. wt-S13 B. m1-S13 C. m2-S13 D. S13/G4 chimera.
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pone-0041124-g002: Images of the plaques (8 h, 37°C, 10 cm Petri dish).A. wt-S13 B. m1-S13 C. m2-S13 D. S13/G4 chimera.

Mentions: Because of S13 sequence restoring a PstI site at each end, the PCR productions of full-length genomes were circularized by ligation with T4 ligase using recommended conditions. To assay the infectivity of circular molecules, each ligation product was electroporated into E.coli C cells, which was the standard laboratory host of S13 phage. Plates were incubated overnight at 37°C and phage plaques were visualized after that. Plaques of the synthetic phages on E. coli strain C on agar plate were 1 or 2 mm in diameter. They formed small, clear, round plaques and some merge together to form a mass (Fig. 2).


Whole-genome synthesis and characterization of viable S13-like bacteriophages.

Liu Y, Han Y, Huang W, Duan Y, Mou L, Jiang Z, Fa P, Xie J, Diao R, Chen Y, Ye Y, Yang R, Chen J, Sun X, Li Z, Tang A, Gui Y, Cai Z - PLoS ONE (2012)

Images of the plaques (8 h, 37°C, 10 cm Petri dish).A. wt-S13 B. m1-S13 C. m2-S13 D. S13/G4 chimera.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3399791&req=5

pone-0041124-g002: Images of the plaques (8 h, 37°C, 10 cm Petri dish).A. wt-S13 B. m1-S13 C. m2-S13 D. S13/G4 chimera.
Mentions: Because of S13 sequence restoring a PstI site at each end, the PCR productions of full-length genomes were circularized by ligation with T4 ligase using recommended conditions. To assay the infectivity of circular molecules, each ligation product was electroporated into E.coli C cells, which was the standard laboratory host of S13 phage. Plates were incubated overnight at 37°C and phage plaques were visualized after that. Plaques of the synthetic phages on E. coli strain C on agar plate were 1 or 2 mm in diameter. They formed small, clear, round plaques and some merge together to form a mass (Fig. 2).

Bottom Line: The results of sequencing showed the accuracy of these synthetic genomes.While no phenotypic differences among the variant strains were observed when grown in LB medium with CaCl(2), the S13/G4 chimera was found to be much more sensitive to the absence of calcium and to have a lower adsorption rate under calcium free condition.These results support an evolutional hypothesis which has been proposed that a homologous recombination event involving gene F of quite divergent ancestral lineages should be included in the history of the microvirid family.

View Article: PubMed Central - PubMed

Affiliation: Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Institute of Urology, Peking University Shenzhen Hospital, Shenzhen PKU-HKUST Medical Center, Shenzhen, China.

ABSTRACT

Background: Unprecedented progresses in high-throughput DNA sequencing and de novo gene synthesis technologies have allowed us to create living organisms in the absence of natural template.

Methodology/principal findings: The sequence of wild-type S13 phage genome was downloaded from GenBank. Two synonymous mutations were introduced into wt-S13 genome to generate m1-S13 genome. Another mutant, m2-S13 genome, was obtained by engineering two nonsynonymous mutations in the capsid protein coding region of wt-S13 genome. A chimeric phage genome was designed by replacing the F capsid protein open reading frame (ORF) from phage S13 with the F capsid protein ORF from phage G4. The whole genomes of all four phages were assembled from a series of chemically synthesized short overlapping oligonucleotides. The linear synthesized genomes were circularized and electroporated into E.coli C, the standard laboratory host of S13 phage. All four phages were recovered and plaques were visualized. The results of sequencing showed the accuracy of these synthetic genomes. The synthetic phages were capable of lysing their bacterial host and tolerating general environmental conditions. While no phenotypic differences among the variant strains were observed when grown in LB medium with CaCl(2), the S13/G4 chimera was found to be much more sensitive to the absence of calcium and to have a lower adsorption rate under calcium free condition.

Conclusions/significance: The bacteriophage S13 and its variants can be chemically synthesized. The major capsid gene of phage G4 is functional in the phage S13 life cycle. These results support an evolutional hypothesis which has been proposed that a homologous recombination event involving gene F of quite divergent ancestral lineages should be included in the history of the microvirid family.

Show MeSH
Related in: MedlinePlus