Today

1
Today

• Assignments
• Collect Pre-lab 8, Worksheet for Ex. 7
• Return 1st submission Bacterial Growth Report
• Exercise 8
• Bacterial Transformation Experiment
• Exercise 7
• Discuss Phage One-step Growth Results- turn in
  work sheet (filled out during lab)
• Work on Environmental Isolate

2
Bacterial Growth Paper

• NO personal pronouns!!!!!!!!!!!!
• I, we, our, etc.
• I will take off lots of points for even one
  personal pronoun!
• Where to put your citations in text
• If you use info from a single source in several
  consecutive sentences, you put the citation once,
  at the end of the info.
• Ex. You would only need to cite the lab manual at
  the end of each paragraph in the Materials and
  Methods

3
Bacterial Growth Papers

• Key words
• Minimal and enriched media
• E. coli
• Optical density
• Generation time

4
Bacterial Growth Paper

• Dont write what is shown in each figure or table
  in the textthe reader should read that in the
  caption of the figure/table.
• Glucose concentration was not 20, 0.5 mL of 20
  glucose was added to the flask.
• YE-P concentration was not 10, 1.5 mL of 10
  YE-P was added.

5
Bacterial Growth Paper

• How to refer readers to your figures/tables in
  you results and discussion
• Make statements, then refer readers to the
  appropriate table or figure.
• Ex. The doubling time in the YE-P enriched
  medium was 37 minutes (Fig. 2).
• Dont say As can be seen in Figure 2.

6
Bacterial Growth Paper

• Media
• Medium
• Mediums

7
Bacterial Growth Paper

• Significance
• In your discussion, why is the increase in the
  growth of the E. coli interesting/significant
• Should tie into the background information in
  your intro.

8
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

9
Bacterial DNA

• Bacterial DNA may be altered by
• Transformation
• Uptake of DNA from the environment

10
Bacterial DNA

• Transduction
• Transfer of DNA via a bacteriophage
• Conjugation
• Transfer of DNA between two touching cells

11
Bacterial Transformation

• Competence 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

12
Bacterial Transformation

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

13
Bacterial Transformation

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

14
Bacterial Transformation Procedure

• Extract DNA from wild-type SD5
• Vortex SD5 culture, and transfer 1.5 mL to
  centrifuge tube
• Centrifuge 1.5 mL of SD5 culture
• Discard supernatant
• Re-suspend pellet in 1.5 mL of lysis solution
• Incubate at 60?C until solution is clear
  (approximately 45 minutes)

15
Bacterial Transformation Procedure

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

16
Bacterial Transformation Procedure

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

17
Bacterial Transformation Procedure

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

18
Bacterial Transformation Procedure

• 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
• Do NOT add any SD6
• Incubate at 37?C for 24-48 hours

19
Bacterial Transformation Procedure

• Finish exercise next week
• Record results
• Complete worksheet

20
Phage 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

21
Plaques
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Worksheet

• Count the number of plaque forming units (pfu)
  for each plate
• 20-200 plaques
• 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

23
Phage Graph
109
Plateau

• Determine Y-axis and X-axis scale
• Label your axes
• Plot points
• Draw best fit lines
• Determine maturation, rise and plateau
• Calculate Burst Size
• plateau (avg)
• maturation (avg)

pfu/mL
Rise
108
Maturation
107
0
10
20
30
40
50
60
24
Next Week

• Assignments
• 2nd submission Bacterial Growth Report Due
• Worksheet 8
• Pre-lab 9
• Exercise 9
• Antibiotics and antimicrobials
• Exercise 8
• Record Results of Bacterial Transformation
  Experiment
• Turn in work sheet (filled out during lab)
• Complete Work on Environmental Isolate