Lab 4: Microbial Genetics Part 1: Bacteriophage viruses

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Lab 4: Microbial Genetics Bacteriophage. Phage cannot replicate on their ownMust infect a host cellOnce inside the host cell, they either remain quiescent (as prophage) or use the cell's replication machinery to produce many copies of themselvesThis replication may or may not lead to destruction of the host cell.

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Lab 4: Microbial Genetics Part 1: Bacteriophage viruses

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1. Lab 4: Microbial Genetics Part 1: Bacteriophage viruses Called “phage” viruses that infect (“eat”) bacteria Relatively simple organisms Basic structure consists of DNA or RNA surrounded by characteristic proteins Vary greatly in shape and size We will focus on DNA-type phage

2. Lab 4: Microbial Genetics Bacteriophage Phage cannot replicate on their own Must infect a host cell Once inside the host cell, they either remain quiescent (as prophage) or use the cell’s replication machinery to produce many copies of themselves This replication may or may not lead to destruction of the host cell

4. Lab 4: Microbial Genetics Bacteriophage Why are phage important? They cause diseases They are useful tools in molecular biology Phage are often used as “replacement vectors”

6. Lab 4: Microbial Genetics Bacteriophage l Infection of E. coli Create the phage “adsorption mix” Mixture of bacteria and phage Plate the adsorption mix Provide media for growth Incubate at 37oC overnight Allow growth to continue ALL phage and bacteria-contaminated waste must be disposed of in designated biohazard waste bins!

7. Lab 4: Microbial Genetics Bacteriophage l Infection of E. coli The adsorption “mix” allows phage to initiate infection process (infect bacteria, E. coli) During the incubation the phage will replicate and lyse the host bacteria The released phage will repeat the process of infection and lysis with neighboring bacteria As time passes, enough lysis will occur so that you will see a clear “plaque” on the plate At the end of the incubation, count the total number of plaques that you see on your plate

8. Picture of phage plaques on a “lawn” of bacteria

9. Lab 4: Microbial Genetics Part 2: Plasmids Segments of DNA that can be found in bacterial cells Exist separate from the bacterial chromosome (extrachromosomal) Circular and relatively small in size Replicate autonomously, and may carry important genes for growth and survival Subcategory of extrachromosomal elements known as episomes.

11. Lab 4: Microbial Genetics Plasmids Bacteria genotypes are defined in part by the media in which they can grow

12. Lab 4: Microbial Genetics Antibiotic Resistance Genes Some plasmids have been genetically engineered to serve as useful vectors for DNA cloning Some carry antibiotic resistance genes (e.g., ampR)

13. Lab 4: Microbial Genetics lacZ Gene Other useful genes (e.g., lacZ)

14. Lab 4: Microbial Genetics Genotyping E. coli Using Selective Media Work in pairs Obtain E. coli strains 1 and 2 Streak a small amount of each strain onto each of three types of selective media

15. Lab 4: Microbial Genetics Genotyping E. coli Using Selective Media Label your section of the plate with your name Bacteria plates will be incubated overnight at 37oC Look at plates later to see if bacteria grew on media Note the appearance of the bacteria Using this data, determine genotypes for the two E. coli strains

16. Lab 4: Microbial Genetics Schedule Lab introduction Create and plate phage adsorption mix Plate bacterial cultures on different plate media Put plates in incubator (370C, overnight) Clean up Go home! Look at plates as soon as possible, but definitely before your next lab (lab summaries are due at BEGINNING of next lab session)

17. Lab 4: Microbial Genetics Lab Summary Purpose Materials and Methods Results Phage plating count or estimate of number of plaques answer questions in lab manual under “phage cultures”, p. 20 Bacteria genotyping results explain reasoning used to describe bacterial genotypes answer questions in lab manual under “bacterial cultures”, p. 20

18. Lab 4: Microbial Genetics Clean Up Be sure that ALL phage and bacteria-contaminated waste has been placed in the designated biohazardous waste containers! Pipette tips, plastic tubes, plastic loops All uncontaminated waste should be disposed of in the regular trash bins ONLY If you are unsure about where to dispose of something, please ask the TA before doing so!!!!!

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