Lab 4: Microbial Genetics Part 1: Bacteriophage viruses

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Lab 4 Microbial GeneticsPart 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

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Lab 4 Microbial GeneticsBacteriophage

• 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 cells
  replication machinery to produce many copies of
  themselves
• This replication may or may not lead to
  destruction of the host cell

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Phage life cycles 1. Lytic pathway New
phage produced that can go off and infect other
cells (exponential growth phase) 2. Lysogenic
pathway Phage genetic material integrates into
host cell chromosome and is replicated as host
divides (quiescent phase)
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Lab 4 Microbial GeneticsBacteriophage

• Why are phage important?
• They cause diseases
• They are useful tools in molecular biology
• Phage are often used as replacement vectors

- Part of their genome DNA is removed and
replaced with other DNA of interest (e.g.,
segments of human DNA)
Recombinant phage can be maintained in solution
(in the laboratory) or they can be grown by
infecting host bacteria
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• bacteriophage
• as a replacement
• vector

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Lab 4 Microbial GeneticsBacteriophage 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!

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Lab 4 Microbial GeneticsBacteriophage 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

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Picture of phage plaques on a lawn of bacteria
plaque
Each plaque represents a clonal population of
phage particles that grew from a single infection
of a bacterial cell at that point on the
plate Each plaque contains a large number of
identical phage particles
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Lab 4 Microbial GeneticsPart 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.

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Plasmids are relatively small circular,
extrachromosomal DNA that naturally occur
in bacteria
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Lab 4 Microbial GeneticsPlasmids

• Bacteria genotypes are defined in part by the
  media in which they can grow

e.g., tet - bacteria cannot grow on media
containing tetracycline, whereas tet R
bacteria can grow on the same media
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Lab 4 Microbial GeneticsAntibiotic Resistance
Genes

• Some plasmids have been genetically engineered to
  serve as useful vectors for DNA cloning
• Some carry antibiotic resistance genes (e.g.,
  ampR)

- Antibiotics often kill bacteria by interfering
with their ability to synthesize
proteins (ampR gene for resistance to the
antibiotic ampicillin)
This gene produces the enzyme b-lactamase. This
enzyme breaks down ampicillin before it has an
effect on the bacteria
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Lab 4 Microbial GeneticslacZ Gene

• Other useful genes (e.g., lacZ)

- Bacteria with a plasmid containing the lacZ
gene can produce the enzyme b-galactosidase
(genotype lac)
If bacteria carrying this gene are grown on
media containing a compound such as X-gal (a
substrate for b-galactosidase) the X-gal can be
cleaved to produce a blue-colored product
Summary Bacteria containing a functional lac
gene will give rise to blue-colored colonies on
media containing X-gal, while bacteria with a
nonfunctional or disrupted gene (lac -) will give
rise to colorless (white) colonies
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Lab 4 Microbial GeneticsGenotyping 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

1 Growth medium X-gal 2 Growth medium X-gal
ampicillin 3 Growth medium X-gal kanamycin
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Lab 4 Microbial GeneticsGenotyping 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

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Lab 4 Microbial GeneticsSchedule

• 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)

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Lab 4 Microbial GeneticsLab 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

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Lab 4 Microbial GeneticsClean 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!!!!!