Bio210-lab-pGLO

Plasmids

  • Self-replicating extrachromosomal DNA molecules found in bacteria.

  • Distinct from chromosomal DNA in bacteria, which is located within the nucleoid region.

Artificial Plasmid Vectors

  • Key Components:

    • Origin of Replication: Allows for DNA replication within a host cell.

    • Selectable Marker: Such as antibiotic resistance, which helps identify cells that have taken up the plasmid.

    • Polylinker / Cloning Site: Contains multiple restriction sites for the insertion of foreign DNA.

Common Features of Plasmid Vectors

  • Replicator: A region of DNA where replication starts (ori).

  • Selectable Marker: Ensures that the plasmid is maintained in the bacterial population (e.g., antibiotic resistance).

  • Cloning Site: Allows for the insertion of foreign DNA using restriction enzyme recognition sites.

Creation of Recombinant DNA

  • Restriction Fragments: The gene of interest is inserted into cut plasmids to form recombinant DNA molecules.

  • Annealing/Ligation: Process of joining DNA fragments together, which may involve cohesive or blunt ends.

Transformation Process

  • Transformation: Introduces recombinant DNA into competent bacteria, making them permeable to DNA.

  • Competent Bacteria: Bacterial cells that can take up foreign DNA.

  • Recombinant Bacterium: Bacteria that harbor the plasmid containing the gene of interest.

Components of the pGLO Plasmid

  • Origin Region: Essential for replication during bacterial cell division.

  • Arabinose Operon (araC): Regulates the expression of the GFP gene in presence of arabinose.

  • Promoter PBAD: Controls transcription of the genes downstream.

  • Ampicillin Resistance Gene: Provides the bacterial cell with resistance to ampicillin.

  • GFP Gene: Codes for Green Fluorescent Protein, allowing for visualization of expression.

Transformation Techniques

  • Calcium Chloride Method: Cells are treated with calcium ions to enhance DNA uptake.

    • Heat Shock: Enhances the ability of cells to take in DNA.

  • Electroporation: Alternative method that uses an electric field to increase cell permeability.

Selection of Transformed Bacteria

  • After transformation, cells are grown in non-selective media before being plated on antibiotic-containing agar.

  • Only bacteria that have taken up the plasmid survive and grow on the selection plate (LB/Amp).

Plasmid Replication

  • Transformed bacteria replicate, producing numerous copies of the recombinant plasmid.

  • Following growth, plasmid extraction (mini-prep) is performed to isolate the recombinant plasmids.

Nucleic Acid Isolation Techniques

  • First step: Lyse cells using chemicals (e.g., alkaline + detergent) to extract total DNA, separating plasmids from chromosomal DNA.

  • Acidification: Neutralizes the solution, allowing plasmids to re-anneal while chromosomal DNA remains tangled.

  • DNA Precipitation: Use of ethanol to precipitate DNA and separate it from contaminants.

Gel Electrophoresis

  • Used to separate DNA fragments based on size via an electric field.

  • Matrix Types: Agarose for large fragments and polyacrylamide for smaller DNA sizes.

  • Detect bands using staining dyes like Ethidium Bromide, which fluoresce under UV light.

Restriction Enzymes

  • Function: Enzymes that cut DNA at specific sequences and are used to create recombinant DNA.

  • Types: Type I, II, and III enzymes, each with a different mechanism for DNA cleavage.

Visualization and Detection of Nucleic Acids

  • Staining: Ethidium bromide is commonly used to visualize DNA in gels; it intercalates with DNA and fluoresces under UV light.

Conclusion and Future Work

  • Next steps involve running agarose gels to assess DNA digestion and performing protein purification techniques using hydrophobic chromatography.