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:A Fundamental Protein Property, Thermodynamic Stability, Revealed Solely From Large-Scale Measurements of Protein Function
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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.
| S-EPMC1681472 | biostudies-literature
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.
| S-EPMC3764833 | biostudies-literature
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.
| S-EPMC9059553 | biostudies-literature
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.
| S-EPMC8449382 | biostudies-literature
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.
| S-EPMC3873743 | biostudies-literature
Project description:Treatment of T7 DNA ligase with a range of proteases generates two major fragments which are resistant to further digestion. These fragments, of molecular weight 16 and 26 kDa, are derived from the N- and C-termini of the protein, respectively. The presence of ATP or a non-hydrolysable analogue, ADPNP, during limited proteolysis greatly reduces the level of digestion. The N-terminal 16 kDa region of the intact T7 ligase is labelled selectively in the presence of [alpha-32P]ATP, confirming that it contains the active site lysine residue. In common with the intact enzyme, the C-terminal portion of the protein retains the ability to band shift DNA fragments of various lengths, implicating it in DNA binding. It can also inhibit ligation by the intact protein, apparently by competing for target sites on DNA. We conclude that the N-terminal region, which contains the putative active site lysine, plays a role in the transfer of AMP from the enzyme-adenylate complex to the 5'phosphate at the nick site, while the C-terminal 26 kDa fragment appears to position the enzyme at the target site on DNA.
| S-EPMC145956 | biostudies-other
Project description:The lagging-strand DNA polymerase requires an oligoribonucleotide, synthesized by DNA primase, to initiate the synthesis of an Okazaki fragment. In the replication system of bacteriophage T7 both DNA primase and DNA helicase activities are contained within a single protein, the bifunctional gene 4 protein (gp4). Intermolecular interactions between gp4 and T7 DNA polymerase are crucial for the stabilization of the oligoribonucleotide, its transfer to the polymerase, and its extension by DNA polymerase. We have identified conditions necessary to assemble the T7 priming complex and characterized its biophysical properties using fluorescence anisotropy. In order to reveal molecular interactions that occur during delivery of the oligoribonucleotide to DNA polymerase, we have used four genetically altered gp4 to demonstrate that both the RNA polymerase and the zinc-finger domains of DNA primase are involved in the stabilization of the priming complex and in sequence recognition in the DNA template. We find that the helicase domain of gp4 contributes to the stability of the complex by binding to the ssDNA template. The C-terminal tail of gp4 is not required for complex formation.
| S-EPMC3386087 | biostudies-other
Project description:Background:The comparatively small genome, well elucidated functional genomics and rapid life cycle confer T7 bacteriophage with great advantages for bio-application. Genetic manipulation of T7 genome plays a key role in T7 related applications. As one of the important aspects in T7 phage genetic modification, gene knock-in refers to two main approaches including direct genetic manipulation in vitro and recombineering. Neither of these available methods are efficient enough to support the development of innovative applications capitalizing on T7 bio-system and thus there is room for novel strategies that address this issue. Integration mediated by the ?C31 integrase is one of the most robust site-specific recombination systems. ?C31 integrases with enhanced activity and specificity have been developed such that it is ideal to effectuate exogenous DNA knock-in of T7 phage with advanced ?C31 integrase. Methods:Plasmid construction was conducted by routine molecular cloning technology. The engineered T7 bacteriophages were constructed through homologous recombination with corresponding plasmids and the functional T7 phage was designated as T7?G10G11-attB. In the integration reaction, hosts with both executive plasmids (pEXM4) and donor plasmids (pMCBK) were lysed by T7?G10G11-attB. Progenies of T7 phages that integrated with pMCBK were isolated in restrict hosts and validated by sequencing. T7?G10G11-attB capacity limit was explored by another integration reactions with donor plasmids that contain exogenous DNA of various lengths. Results:T7?G10G11-attB exhibits abortive growth in restrictive hosts, and a bacterial attachment site recognized by ?C31 integrase (attB) was confirmed to be present in the T7?G10G11-attB genome via sequencing. The integration reaction demonstrated that plasmids containing the corresponding phage attachment site (attP) could be integrated into the T7?G10G11-attB genome. The candidate recombinant phage was isolated and validated to have integrated exogenous DNA. The maximum capacity of T7?G10G11-attB was explored, and it's found that insertion of exogenous DNA sequences longer than 2 kbp long can be accommodated stably. Conclusion:We advanced and established a novel approach for gene knock-in into the T7 genome using ?C31 integrase.
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