Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Lect-11 - WordPress.com
Document related concepts
Transcript
LECTURE 11: Phage identification and characterization Viro102: Bacteriophages & Phage Therapy 3 Credit hours Atta-ur-Rahman School of Applied Biosciences (ASAB) 13 Bacteriophage families Double stranded DNA, Non-enveloped P2 Double stranded DNA, Enveloped SIRV 1, 2 Rudiviridae Myoviridae T2 Fuselloviridae SSV1 λ Tectiviridae Plasmaviridae TTV1 PRD1 Siphoviridae Lipothrixviridae P22 Corticoviridae Podoviridae Single-stranded DNA Inoviridae M13 & fd PM2 Single stranded RNA Double stranded RNA phi666 MS2 Microviridae ΦX174 Leviviridae Cystoviridae Bacteriophage: ICTV Classification Family Myoviridae Morphology Non-enveloped, contractile tail Nucleic acid Linear dsDNA Siphoviridae Non-enveloped, long non-contractile tail Linear dsDNA Podoviridae Non-enveloped, short non contractile tail Linear dsDNA Tectiviridae Corticoviridae Non-enveloped, isometric Non-enveloped, isometric Lipothrixvirida Enveloped, rod-shaped Linear dsDNA Circular dsDNA Linear dsDNA Bacteriophage: ICTV Classification Plasmaviridae Enveloped, pleomorphic Circular dsDNA Rudiviridae Linear dsDNA Non-enveloped, rod-shaped Fuselloviridae Non-enveloped, lemon-shaped Circular dsDNA Inoviridae Non-enveloped, filamentous Circular ssDNA Microviridae Non-enveloped, isometric Circular ssDNA Leviviridae Non-enveloped, isometric Linear ssRNA Cystoviridae Enveloped, spherical Segmented dsRNA Bacteriophage Characterization • Electron microscopy (Morphological Studies) • Bacteriophage DNA isolation – RFLP – Genome fingerprinting by RAPD analysis • SDS-PAGE analysis of phage proteins • Burst size • Bacteriophage host range Electron Microscopy • Aliquots of a bacteriophage sample obtained by ultracentrifugation were subjected to electron microscopy for morphological analysis. • Purified phage particles were negatively stained with 2% (wt/vol) uranyl acetate, deposited on carbon-coated grids. Isometric heads, visible collars, and shorter contractile tails with terminal base plates, which are characteristics of the family Myoviridae Electron micrographs of L. fallax bacteriophages. (A) R01; (B) R03; (C) R05; (D) R09; (E) R12; (F) R19. Bars Electron Microscopy Electron Microscopy Bacteriophage host range • Phage host range can be done by using the following test method against different host – Spot test – Plaque assay – Streak Assay Bacteriophage host range Spotting can be used as a means of determining a phage's host range. Spot Test Spotting can be used to provide a first approximation of the ability of a phage to lyse or just kill a bacterial strain. This is done such as during phage typing procedures or, at lower phage densities, as a means of phage titering that is less materials intensive than full plate-count assays. Streak Assay Bacteriophage host range Plaque counting Unit (PFU) virulent phage stock and a susceptible host cell culture. 10-fold dilutions of the phage stock are prepared. The procedure requires the use of a Double-Layer Agar (DLA) technique also known as double agar overlay method, in which the hard agar serves as a base layer (to form gel), and a mixture of few phage particles (diluted stock) and a very large number of host cells in a soft agar forms the upper overlay. When the plates are incubated, susceptible E. coli cells multiply rapidly and produce a lawn of confluent growth on the medium. When one phage particle adsorbs to a susceptible cell, penetrates the cell, replicates and release new phage particles which infect other bacteria in the vicinity of the initial host cell. The destroyed cells produce single circular, non turbid areas called plaques in the bacterial lawn, where there is no growth of bacteria. Each plaque represents the lysis of a phage-infected bacterial culture and can be designated as a plaque-forming unit (PFU) and is used to quantitate the number of infective phage particles in the culture. Dyes that stain the living cells are frequently used to enhance the contrast between the plaques and the living cells Bacteriophage DNA isolation • One hundred ml of phage lysate was incubated for 1 h at 37°C after addition of DNase I and RNase A(2 µg/ml). • Incubated • The phages were pellet down by centrifugation • phage pellets • To degrade bacterial DNA and RNA in the lysate, you will use a mix of nucleases (DNase I and RNase A). The capsid of the phage protects its DNA from the nucleases. However, if nucleases are present in later steps of the protocol, the phage DNA may become degraded. Phenol Chloroform extraction • The pellet obtained after centrifugation is dissolved in 100 micro liter autoclaved H20. • Treated with DNase I & incubated for 370C • SDS, proteinase K • Phenol chloroform extraction Phenol chloroform method Phage Genome (Agarose Gel) 23130bp 20-23 kb 9416bp 6557bp 4361bp 2322bp 2027bp 1 2 3 4 Genome fingerprinting by restriction fragment length polymorphism (RFLP) analysis • Purified bacteriophage DNA samples were subjected to restriction enzyme digestion • with AluI, BamHI, EcoRI, HindIII, MboI, RsaI, and Sau3AI • The restriction digests were separated on a 0.8% agarose gel and stained with ethidium bromide. RFLP analysis of phage DNA: EcoRI digestion patterns of Myoviridae bacteriophages. Lane M, 1-kb DNA ladder; lane 1, R03; lane 2, R05; lane 3, R12. Genome fingerprinting by RAPD analysis • The method used for randomly amplified polymorphic DNA (RAPD) analysis • Add primers and template DNA • PCR • Agarose gel Phage A Phage B SDS-PAGE analysis of phage proteins. • Bacteriophage structural proteins were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS PAGE). • Crude extract • Specific protein extraction SDS-PAGE patterns of phage structural proteins. Lanes M, molecular weight markers; lane 1, Siphoviridae phage R01; lane 2, Myoviridae phage R03; lane 3, Siphoviridae phage R09. Phage Burst Size, Latent period R L 1400 P Bacteriophage Count pfu/ml 1200 1000 800 801 LK1/ cell 600 24 min. 400 200 0 0 3 6 9 12 15 18 21 24 27 30 33 36 Time (min) One step growth curve showing the latent period (24 min) and the average burst size (801 vira particles per host cell). Latent time and burst size of phage LK1 were inferred from the curve with a triphasic pattern. L: latent phase;R: rise phase; P: plateau phase. Measuring bacteriophage burst size Does each phage-infected bacterial cell release the same number of phage or does the number vary from cell to cell? If it varies, can we define the factors involved in this variation? To measure an average burst size lets perform this online experiment http://virtuallaboratory.colorado.edu/BioFunSupport/labs/OnMutation/section_04.html http://virtuallaboratory.colorado.edu/BioFunSupport/labs/OnMutation/flash/vCOUNT.html Thanks!!
