Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
TEAM 1 Investigation of bacteriophages of the bird pathogen, Bordetella avium Introduction What is Bordetella avium? • Bacteria that causes upper respiratory disease bordetellosis in avian species (chicken, turkey, etc.) • Gram negative, non-fermentable, aerobic and motile • Infects commercially grown turkeys throughout the world Hosts for B. avium SO …what? B. avium infections result in severe economic losses in all poultry-producing regions of the world. What is a bacteriophage? A bacteriophage is a virus that solely infects bacteria. What is a virus? Obligate parasite, cannot replicate itself Needs a host cell for survival Structure of Bacteriophage •Phage head: composed of coat protein and genome in the core •Genome: DNA codes for enzymes and proteins necessary to replicate more viruses •Tail Sheath: DNA travels from head to bacteria through sheath •Tail fiber: helps anchor the phage on the cell membrane Life cycles of a Temperate Phage Two life cycles: Lytic cycle – viruses lyse the cell after replicating in the host cell Lysogenic cycle – viral DNA integrates into host cell DNA to replicate, but no new viruses are synthesized http://www.msu.edu/course/lbs/145/s02/graphics/campbell_18.5.gif Ba1-1 and Ba1-2 • First identified as Ba1 • After sequencing, two phage chromosomes were found Ba1-1, Ba1-2 • Under electron microscope, look exactly the same SEM of Phage About 2/3 of the chromosomes are the same Ends- enzymes Center- structure Goal To determine which phage(s) were in each B. avium strain Methods and Materials Behavior in Live Bacteria How did we check for active phage? Spontaneous lysis 197N infection Spontaneous Lysis Grow single colonies of bacteria in Brain Heart Infusion (BHI) broth Add culture to melted BHI top agar and spread over a BHI plate Incubate at 30°C for 18-24 hours, examine for plaques Cartoon of a plate with plaques plaque bacteria Testing bacterial strains for phage that infect 197N Add two drops chloroform to cultures to kill bacteria, leaving only phage Combine this culture with 197N culture, plate and incubate Examine for plaques Polymerase Chain Reaction Goal: amplification of a small amount of DNA Ingredients: template DNA, primers, nucleotides, and thermostable Taq Polymerase Polymerase Chain Reaction Ingredients run through a series of heating and cooling cycles Denaturation – DNA separated into two strands Annealing – Primers attach Polymerization – free bases attach Our Primers Beginning primers unique to Ba11 and Ba1-2 (left) Recombinase primers from Ba1-1 and Ba1-2 (right) Tail Fiber primer identical in both strains (middle) Gel Electrophoresis Method used to analyze PCR PCR product injected into gel wells Apply electrical field DNA travels from the negative electrode to the positive Travels through gel based on size Gel Electrophoresis Results read as dark lines in the gel Fragment size read against a 1 kb (1000 base pair) ladder Ethidium Bromide makes discrete bands visible under UV Agarose Gel & Gel Electrophoresis Results Results for spontaneous lysis and infection of 197 N Strain Spontaneous Lysis Infection of 197 N Wampler 0 0 + + 0 0 197 N JBBA Gel of Repressor Primer Results for PCR Strain Unk-1 Rec 1 Tail fiber Rep-2 Rec 2 Wampler + + + + 0 197 N 0 0 + + 0 G24 0 0 0 0 0 Discussion Discussion Interpretation of results Effectiveness of methods Future applications and extensions of our work Reasons for Tests Behavior of phage in strain of B.avium Spontaneous lysis test 197N infection test Which part of phage DNA in bacterial DNA? PCR & gel electrophoresis using primers that amplified pieces of DNA from different phages Spontaneous Lysis Which strains made plaques? What does that mean? What if there were no plaques? T4 Wampler 239 Phage present and lytic Phage absent Phage highly lysogenic Not enough phage Infection of 197N Which strains made plaques? What does that mean? What if there were no plaques? T4 Wampler 239 Ba011 Ba177 DBL260 ATCC Phage present and able to infect 197N Phage absent Phage unable to infect 197N Not enough phage The plates don’t tell… If plaques… which phage active in that strain? If no plaques… does the bacterial strain still contain some phage DNA? Which pieces? T4 and Wampler •Positive in four primers •Spontaneous Lysis, Infection results also positive •Probably contain both phages D4, D10, and D27 All PCR primers yielded positive Probably contains phages Spontaneous Lysis, Infection results negative Could be phage debris May be in lysogenic cycle Conditions may not be right for lysis G24 All PCR Primers yield negative Spontaneous Lysis, Infection negative Probably does not contain phages 197N and ATCC Some negative, some positive ATCC—Unk-1, Rec 1, TF positive Infection positive Probably Ba 1-1, not Ba 1-2 197N—Rep-2, TF positive Infection positive Probably Ba 1-2,not Ba 1-1 What’s next? Experiments only the beginning More PCR Results not perfect, more PCR means more accuracy Different primers More Spontaneous Lysis and Infection Don’t fully understand conditions for lysis More Future Projects Compare DNA/Genes of Ba 1-1 and 1-2 Similar genes code for proteins common in both phages—head, tail fiber… Different genes will code for differences—enzymes Researchers can discover what makes the strains different Acknowledgements Thank you to… Dr. Temple Holly Kuzmiak Kelly Prescott Octawia Wojcik Drew Biology Department Dr. Miyamoto