T7 Bacteriophage WW-II phage display
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA175232 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR569005_1.fastq.gz | Fastqsanger.gz | |||
| SRR569005_2.fastq.gz | Fastqsanger.gz | |||
| SRR569006_1.fastq.gz | Fastqsanger.gz | |||
| SRR569006_2.fastq.gz | Fastqsanger.gz | |||
| SRR569007_1.fastq.gz | Fastqsanger.gz |
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Project description:Natural biological systems are selected by evolution to continue to exist and evolve. Evolution likely gives rise to complicated systems that are difficult to understand and manipulate. Here, we redesign the genome of a natural biological system, bacteriophage T7, in order to specify an engineered surrogate that, if viable, would be easier to study and extend. Our initial design goals were to physically separate and enable unique manipulation of primary genetic elements. Implicit in our design are the hypotheses that overlapping genetic elements are, in aggregate, nonessential for T7 viability and that our models for the functions encoded by elements are sufficient. To test our initial design, we replaced the left 11,515 base pairs (bp) of the 39,937 bp wild-type genome with 12,179 bp of engineered DNA. The resulting chimeric genome encodes a viable bacteriophage that appears to maintain key features of the original while being simpler to model and easier to manipulate. The viability of our initial design suggests that the genomes encoding natural biological systems can be systematically redesigned and built anew in service of scientific understanding or human intention.
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Project description:Phage T7 discontinues host gene expression by translational and transcriptional control mechanisms. Translational control is exerted by the T7 translational-repressor. This protein inhibits the synthesis of beta-galactosidase (EC 3.2.1.23) in vivo and in vitro and the synthesis of the T3 enzyme S-adenosylmethioninehydrolase (EC 3.3.1.-). The translational-repressor does not interfere with T7-specific enzyme synthesis. The T7 translational-repressor purifies with the initiation factors. The repressor interacts with the initiation of translation of host enzymes. The translational-repressor gene is close to the promotor for RNA polymerase of Escherichia coli.
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Project description:Most bacterial viruses need a specialized machinery, called "tail," to inject their genomes inside the bacterial cytoplasm without disrupting the cellular integrity. Bacteriophage T7 is a well characterized member of the Podoviridae family infecting Escherichia coli, and it has a short noncontractile tail that assembles sequentially on the viral head after DNA packaging. The T7 tail is a complex of around 2.7 MDa composed of at least four proteins as follows: the connector (gene product 8, gp8), the tail tubular proteins gp11 and gp12, and the fibers (gp17). Using cryo-electron microscopy and single particle image reconstruction techniques, we have determined the precise topology of the tail proteins by comparing the structure of the T7 tail extracted from viruses and a complex formed by recombinant gp8, gp11, and gp12 proteins. Furthermore, the order of assembly of the structural components within the complex was deduced from interaction assays with cloned and purified tail proteins. The existence of common folds among similar tail proteins allowed us to obtain pseudo-atomic threaded models of gp8 (connector) and gp11 (gatekeeper) proteins, which were docked into the corresponding cryo-EM volumes of the tail complex. This pseudo-atomic model of the connector-gatekeeper interaction revealed the existence of a common molecular architecture among viruses belonging to the three tailed bacteriophage families, strongly suggesting that a common molecular mechanism has been favored during evolution to coordinate the transition between DNA packaging and tail assembly.
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Project description:Here, we describe the complete genome sequence of the Escherichia coli bacteriophage vB_EcoP_PR_Kaz2018, isolated from a water sample. vB_EcoP_PR_Kaz2018 is a linear double-stranded DNA T7-like podophage with a genome of 39,704 bp containing 45 predicted open reading frames (ORFs).
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Project description:Transcriptional repressors play an important role in regulating phage life cycle. Here, we examine how synthetic transcription repressors can be used in bacteriophage T7 to create a dynamic, controllable infectivity switch. We engineered T7 phage by replacing a large region of the early phage genome with different combinations of ligand-responsive promoters and ribosome binding sites (RBS) designed to control the phage RNA polymerase, gp1. Phages with engineered infectivity switch are fully viable at levels comparable to wildtype T7, when not repressed, indicating the phage can be engineered without loss of fitness. The most effective switch used a TetR-responsive promoter and an attenuated RBS, resulting in a 2-fold increase in latent period and a 10-fold decrease in phage titer when repressed. Phage activity can be further tuned using different inducer concentrations. Our study provides a proof of concept for how a simple synthetic circuit introduced into the phage genome enables user control over phage infectivity.
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Project description:Many tailed bacteriophages assemble ejection proteins and a portal-tail complex at a unique vertex of the capsid. The ejection proteins form a transenvelope channel extending the portal-tail channel for the delivery of genomic DNA in cell infection. Here, we report the structure of the mature bacteriophage T7, including the ejection proteins, as well as the structures of the full and empty T7 particles in complex with their cell receptor lipopolysaccharide. Our near-atomic-resolution reconstruction shows that the ejection proteins in the mature T7 assemble into a core, which comprises a fourfold gene product 16 (gp16) ring, an eightfold gp15 ring, and a putative eightfold gp14 ring. The gp15 and gp16 are mainly composed of helix bundles, and gp16 harbors a lytic transglycosylase domain for degrading the bacterial peptidoglycan layer. When interacting with the lipopolysaccharide, the T7 tail nozzle opens. Six copies of gp14 anchor to the tail nozzle, extending the nozzle across the lipopolysaccharide lipid bilayer. The structures of gp15 and gp16 in the mature T7 suggest that they should undergo remarkable conformational changes to form the transenvelope channel. Hydrophobic α-helices were observed in gp16 but not in gp15, suggesting that gp15 forms the channel in the hydrophilic periplasm and gp16 forms the channel in the cytoplasmic membrane.
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Project description:Adsorption and genome ejection are fundamental to the bacteriophage life cycle, yet their molecular mechanisms are not well understood. We used cryo-electron tomography to capture T7 virions at successive stages of infection of Escherichia coli minicells at ~4-nm resolution. The six phage tail fibers were folded against the capsid, extending and orienting symmetrically only after productive adsorption to the host cell surface. Receptor binding by the tail triggered conformational changes resulting in the insertion of an extended tail, which functions as the DNA ejection conduit into the cell cytoplasm. After ejection, the extended phage tail collapsed or disassembled, which allowed resealing of the infected cell membrane. These structural studies provide a detailed series of intermediates during phage infection.
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