BACTERIAL TRANSFORMATION Flashcards
A process in which bacteria take up foreign DNA from their environment.
Bacterial Transformation
Importance of Bacterial Transformation
Used in genetic engineering, research, and biotechnology applications
Common Method of Bacterial Transformation
Heat-shock transformation using plasmid DNA
Objectives of the Bacterial Transformation Experiment
1 Transform Escherichia coli with plasmid DNA using a heat-shock method.
2 Demonstrate competency induction in bacterial cells.
3 Observe antibiotic resistance as an indicator of successful transformation.
Materials and Reagents of the Bacterial Transformation Experiment
1 Competent E. coli (ATCC 25922)
2 Plasmid DNA (antibiotic-resistant)
3 LB broth (Luria-Bertani broth) & LB agar with antibiotic (e.g., ampicillin)
4 Ice, 42°C water bath, 37°C incubator
5 Sterile micropipettes, glass beads, sterile loop
Preparation of Competent Cells
1 Grow E. coli in LB broth overnight at 37°C.
2 Centrifuge at 4000 x g for 5 minutes and discard supernatant.
3 Resuspend the pellet in 10 mL of cold 0.1 M CaCl₂ solution.
4 Incubate on ice for 5 minutes to enhance competency.
5 Repeat centrifugation and resuspend in 0.1 M CaCl₂.
6 Aliquot 100 μL into sterile microcentrifuge tubes.
7 Store at -80°C for future use.
Preparation of 0.1 M CaCl₂ Solution
1 Dissolve 1.47 g of CaCl₂·2H₂O in 100 mL deionized water.
2 Keep on ice before use.
3 Maintain pH at 6.5-7.0 if necessary.
LB Broth Preparation
1 Weigh 10 g tryptone, 5 g yeast extract, and 10 g NaCl.
2 Dissolve in 1 L deionized water.
3 Adjust pH to 7.0 using NaOH.
4 Autoclave at 121°C for 15-20 minutes.
LB Agar Preparation
1 Prepare LB broth as above.
2 Add 7 g agar to 500 mL LB broth.
3 Autoclave at 121°C for 15-20 minutes.
4 Cool to 50-55°C before pouring plates.
Ampicillin Preparation
1 Dissolve 500 mg ampicillin in 5 mL distilled water (100 mg/mL stock solution).
2 Filter sterilize using a 0.22 μm filter.
3 Store at -20°C.
4 Add 500 μL to 500 mL LB agar cooled to 50°C before pouring plates.
Step-by-Step Transformation Procedure
1 Thaw competent E. coli cells on ice.
2 Add 1-5 μL of plasmid DNA to the cells.
3 Incubate on ice for 15-30 minutes.
4 Perform heat shock at 42°C for 30 seconds.
5 Quickly return to ice for 2-5 minutes.
6 Add 250-500 μL LB broth and incubate at 37°C for 30-60 minutes.
7 Plate onto LB agar with antibiotic and incubate overnight.
__ = successful transformation
Growth on LB agar with antibiotic
No growth
failed transformation or improper antibiotic resistance selection.
Possible controls:
Non-transformed E. coli plate
Factors Affecting Transformation Efficiency
1 DNA concentration & quality.
2 Competency of E. coli cells.
3 Heat-shock duration and temperature.
4 Recovery time and antibiotic selection.
Applications of Bacterial Transformation
1 Genetic engineering & cloning.
2 Protein production (e.g., insulin synthesis).
3 Antibiotic resistance studies.
4 Bioremediation & synthetic biology.
Function: Host bacteria that will take up foreign plasmid DNA.
Special characteristic: Rendered competent using _ to allow DNA uptake.
Competent E. coli (ATCC 25922)
- CaCl₂
Function: Circular DNA carrying genes for antibiotic resistance and other selectable markers.
Importance: Provides a selectable trait ensuring only _ survive.
Plasmid DNA (antibiotic-resistant)
- transformed cells
Function: Nutrient-rich medium that supports bacterial growth.
Contains ,,_ for cell nutrition.
LB Broth (Luria-Bertani broth)
- tryptone, yeast extract, and NaCl
Function: Solid medium used for plating transformed bacteria.
Importance: Selective pressure ensures only _ grow.
LB Agar with Antibiotic (e.g., ampicillin)
- transformed bacteria
Function: Increases the competency of bacterial cells by making the cell membrane more permeable.
Importance: Helps facilitate _ uptake.
0.1 M CaCl₂ Solution
- DNA
Function: Maintains competency of _cells before transformation.
Importance: Prevents early , preserving.
Ice
- E. coli
- heat shock response
- cell viability
Function: Provides the necessary heat shock to induce DNA uptake.
Importance: Creates a _ allowing plasmid DNA to enter cells.
42°C Water Bath
thermal imbalance
Function: Maintains optimal growth temperature for E. coli.
Importance: Allows bacteria to recover and express _.
37°C Incubator
- antibiotic resistance genes
Function: Used for precise transfer of small liquid volumes.
Importance: Prevents contamination and ensures accurate measurements.
Sterile Micropipettes & Tips
Function: Used to spread bacterial culture evenly on agar plates.
Importance: Ensures uniform colony distribution for analysis.
Glass Beads or Sterile Loop
