Lab 4: Microbial Genetics Schedule

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Lab 4 Microbial GeneticsSchedule

• Lab introduction
• Review Microbiology Techniques (Pipettes, Tips,
  Centrifuge)
• Create and plate phage adsorption mix
• Bacterial genotyping exercise
• Clean up
• View results of bacterial plates next Tuesday
  between 2 and 4 pm.

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Lab 4 Microbial GeneticsVocabulary

• Vector General term for a DNA molecule that is
  used as a vehicle to introduce foreign DNA into a
  host cell.

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Lab 4 Microbial GeneticsVocabulary

• Phage A virus, it is one kind of vector.
• Bacteriophage a phage (virus) that infects
  bacteria

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Lab 4 Microbial GeneticsVocabulary

• Plasmid circular DNA molecules found in many
  bacteria. These are present in addition to the
  regular bacterial genome and are
  self-replicating.

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Lab 4 Microbial GeneticsBacteriophage l
Infection of E. coli

• Create the phage adsorption mix
• Plate the adsorption mix
• Invert the plate and incubate at 37oC overnight
• ALL phage and bacteria-contaminated waste must be
  disposed of in designated biohazard waste bins!

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Lab 4 Microbial GeneticsBacteriophage

• Called phage, or viruses that infect bacteria
• Simple organisms
• Basic structure consists of DNA or RNA surrounded
  by a protein coat
• 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
• 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 cycle Lytic vs. Lysogenic pathways
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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 DNA is removed and replaced with
other DNA of interest (e.g., human DNA)
- This recombinant DNA is repackaged into phage
phage infect host bacteria
- Phage use bacteria to replicate, therefore
replicating the DNA of interest
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• bacteriophage
• as a replacement
• vector

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Lab 4 Microbial GeneticsBacteriophage l
Infection of E. coli

• Infected E. coli with bacteriophage l
  (adsorption)
• While the plate is incubating, phage will
  replicate and lyse the host bacteria
• The released phage will then 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 (spot) on the media
  plate
• At the end of the plate incubation, you will
  count the total number of plaques that you see on
  your plate

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Lab 4 Microbial GeneticsExtrachromosomal DNA in
Prokaryotes

• Extrachromosomal DNA is often used by bacteria to
  carry important genes for growth and survival
• Called plasmids, they are circular and relatively
  small in size
• Plasmids can occur naturally or be genetically
  engineered and inserted into bacteria
• Some plasmids can integrate into the bacterial
  chromosome, while others exist independently from
  the chromosome

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Plasmids are relatively small circular,
extrachromosomal DNA that occur in bacteria
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Lab 4 Microbial GeneticsExtrachromosomal DNA in
Prokaryotes

• What types of genes do these DNAs contain?
• Antibiotic resistance
• Genes allowing them to metabolize other compounds
  present in their growth media
• Bacteria genotypes are defined 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 (because it is
resistant to tetracycline)
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Lab 4 Microbial GeneticsAntibiotic Resistance
Genes

• Plasmids are genetically engineered to serve as
  useful vectors for DNA cloning
• Antibiotic resistance (e.g., ampR)

- Antibiotics often kill bacteria by interfering
with their ability to synthesize proteins

• ampR bacteria
• -Do not produce ampicillin, they are Resistant to
  it
• Do produce the enzyme b-lactamase
• b-lactamase breaks down ampicillin before it has
  an effect on the bacteria

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Lab 4 Microbial GeneticsOther useful genes -
lacZ
- When bacteria have a plasmid containing the
lacZ gene, they will produce b-galactosidase
(genotype lac )
- X-gal is a substrate for b-galactosidase

• If b-galactosidase producing bacteria (lac) are
  grown on media plates containing X-gal,
• X-gal will be cleaved to produce a blue-colored
  product

- lac bacteria will give rise to blue-colored
colonies on media plates containing X-gal, -
lac - bacteria will not make b-galactosidase, not
cleave X- gal, and will then give rise to 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
Note ampicillin and kanamycin are antibiotics
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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 at 37oC
• Look at plates on Tuesday 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 GeneticsLab Summary

• Results (in tables)
• Phage plating results
• Bacteria genotyping results
• Conclusions
• Comment on phage plating results
• Explain reasoning used to determine bacterial
  genotypes

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Time/date stamp
When you are returning assignments to ME
Drop box
375L TA mailboxes
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When I am returning assignments to YOU
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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 in
  desktop disposal bin
• Gloves in large container on the floor near the
  back of the room
• All uncontaminated waste should be disposed of in
  the regular trash bins only

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Lab 4 Microbial GeneticsClean Up

• When Must Personal Protective Equipment Be Worn?
• When there is active manipulationof cultures
  happening anywhere in the lab
• that is, anytime anyone has an open or exposed
  vial of bacteria, bacterial-contaminated tubes,
  culture plates, etc.