Exercise 8 Bacterial Transformation

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Exercise 8Bacterial Transformation

• Bacterial Transformation
• Phage One-Step Growth Worksheet
• Environmental Isolate
• MARTZ

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The Background Information
Bacterial DNA

• The Chromosome
• Necessary for survival
• Information coded in genes
• Plasmids
• Not essential to survival, but may enhance
  survival
• Antibiotic resistance
• Ability to degrade a substrate for uptake into
  the cell

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Background Information
Bacterial genomes may be altered through

• Transformation
• Uptake of DNA from the environment
• Transduction
• Transfer of DNA via a bacteriophage
• Conjugation
• Transfer of DNA between two touching cells

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Transformation, Transduction, Conjugation
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Transformation, Transduction, Conjugation
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Transformation, Transduction, Conjugation
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Transduction
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Transduction
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Conjugation
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Bacterial Transformation

• Transformation is the uptake of DNA from the
  environment
• May be spliced into bacterial chromosome
• May become a plasmid

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Bacterial Transformation

• Competance is the ability of a bacterium to take
  up DNA from the environment
• Escherichia coli must be treated to become
  competent
• Chemical, physical
• DNA coated beads air-blasted into cells
• Acinetobacter calcoaceticus are always competent

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Bacterial TransformationPurpose

• Transform mutant to wild-type
• Acinetobacter calcoaceticus strain SD6
• Tryptophan auxotroph mutant
• Acinetobacter calcoaceticus strain SD5
• Wild-type

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Bacterial Transformation

• Acinetobacter calcoaceticus
• Donor Wildtype strain SD5
• Recipent Mutant strain SD6

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Bacterial TransformationProcedure

• Acetate minimal medium plates
• No tryptophan
• Extract DNA from wild-type SD5
• Apply DNA to a spread plate with SD6
• Apply DNA to a control plate
• Incubate

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Bacterial TransformationProcedure

• Extract DNA from wild-type SD5
• Vortex SD5 culture
• Centrifuge 1.5 mL of SD5 culture
• Discard supernatant
• Resuspend pellet in 1.5 mL of lysis solution
• Incubate at 60?C until solution is clear
  (approximately 45 minutes)

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Bacterial TransformationProcedure

• Incubate SD5 cells in lysis solution at 60?C for
  about 45 minutes

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Bacterial TransformationProcedure

• Prepare one transformation plate
• Prepare one control plate
• Draw circles on the bottom for DNA extraction and
  dilutions
• Undiluted U
• 110
• 1100

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Bacterial TransformationProcedure

• Prepare serial dilutions of extracted SD5 DNA (or
  lysate) in sterile water
• Use sterile technique
• Final dilutions of 110 and 1100
• 110
• 0.5 mL lysate
• 4.5 ml sterile water

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Bacterial TransformationProcedure

• Transformation plate
• Make one spread plate with mutant SD6
• Apply SD5 DNA dilutions to transformation plate
• Control plate
• Apply SD5 DNA dilutions to control plate
• Incubate at 37?C for 24-48 hours

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Bacterial Transformation

• Finish exercise next week
• Record results
• Complete worksheet

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Phage One-Step GrowthPhage Plaque Assay

• Last week
• Infect E. coli strain B with T4 phage
• Remove samples over time
• Plate diluted samples with E. coli lawn cells
• Incubate at 37?C for 24-48 hours
• Today
• Count plaque forming units
• Complete bacteriophage worksheet

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Phage One-Step GrowthExercise 7 Worksheet

• Count the number of plaque forming units (pfu)
  for each plate
• Calculate pfu/mL
• Plot data onto 3-cycle semi-log paper
• Calculate burst size
• Divide the average pfu/mL at the plateau by the
  average pfu/mL during the maturation period

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Phage One-Step Growth
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Plateau
Plotting the data

• Determine Y-axis
• and X-axis scale

OD550
Rise

• Label your axes

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• Plot points

• Draw best fit lines

Maturation

• Determine maturation,
• rise and plateau

Calculate Burst Size plateau (avg) maturation
(avg)
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0
10
20
30
40
50
60
Time(min)