Bacterial Transformation

Bacterial Transformation Process

• Objective: To produce a substantial amount of protein using bacterial cells.
• Steps:
  1. Identify the gene responsible for producing the desired protein.
  2. Insert the gene into a bacterial plasmid.
  3. Introduce the plasmid into bacterial cells.
  4. Cultivate the bacteria to create a large population.
  5. Extract and purify the protein produced by the bacteria.

Methods for Gene Delivery into Bacteria

• Plasmid Creation:
  • Plasmids are circular DNA molecules that can replicate independently.
  • Oftentimes, natural bacterial plasmids are modified to carry specific genes.
• Bacterial Transformation:
  • The process by which bacteria take up foreign DNA (such as plasmids).

Components and Features of Plasmids

• Origin of Replication:
  • Allows for replication of the plasmid within bacterial cells.
• Gene of Interest:
  • Contains the gene for the protein we wish to produce or other functional traits.
• Antibiotic Resistance Gene:
  • Enables transformed bacteria to survive in the presence of antibiotics, allowing for selection of successful transformations.
  • Purpose: Ensure only transformed bacteria survive; other non-transformed bacteria are eliminated.

Example of a Protein: Green Fluorescent Protein (GFP)

• Originates from the jellyfish Aequorea victoria.
• Exhibits fluorescence under UV light.
• pGLO Plasmid: A specific plasmid engineered to include the GFP gene.

Bacterial Transformation Procedure

1. Start with E. coli (common bacteria used in labs).
2. Add plasmid containing the gene of interest.
3. Prepare Transformation Solution (CaCl2):
   • Neutralizes the negative charge of DNA, assisting in the uptake by bacteria.
4. Heat Shock Treatment:
   • Involves rapid temperature changes to increase the permeability of bacteria’s cell membranes.
5. Recovery on Ice:
   • Allows bacteria to stabilize post-heat shock (2 mins).
6. Add LB Broth:
   • Provides nutrients, enabling growth (10 mins).

Transformation Steps Breakdown

• CaCl2 Solution:
  • Shields negative charges on DNA for easier uptake.
• Pre-Incubation on Ice:
  • Slows membrane fluidity for effective heat shock.
• Heat Shock:
  • Enhances cell membrane permeability, allowing plasmids to enter cells.
• Post-Heat Shock on Ice:
  • Restores the integrity of the cell membrane.
• Incubation at Room Temperature:
  • Allows expression of the antibiotic resistance gene (beta-lactamase).
• Spreading on Selective Media:
  • Plates are infused with ampicillin to select for only transformed bacteria.

Selective Media and Growth Conditions

• Ampicillin: A key antibiotic used in media to select for transformed bacteria.
  • Transformed bacteria produce beta-lactamase to deactivate ampicillin, allowing them to grow.
  • Non-transformed bacteria cannot survive on ampicillin mediums.
• LB Broth:
  • Nutrient-rich growth medium (Lysogeny Broth or Luria-Bertani):
  • Contains carbohydrates, amino acids, nucleotides, salts, and vitamins necessary for bacterial growth.

Gene Expression Regulation in Bacteria

• araC Protein:
  • Regulatory protein that modulates gene expression in the presence of arabinose (a sugar).
  • Without Arabinose: araC blocks RNA polymerase, preventing GFP transcription.
  • With Arabinose: araC alters shape, enabling gene transcription for GFP, leading to green fluorescence.