U3L8 - Bioengeneering

Biotechnology

  • Definition of Biotechnology

    • The use of living organisms, or substances from living organisms, to develop agricultural, medicinal, or environmental products or processes.

Examples of Biotechnology

  • Selective Breeding

    • Humans breed plants or animals over multiple generations to select for desirable traits.

    • Process:

    • Animals or plants with desirable traits are chosen for breeding in the next generation until the optimal organism is created.

  • Domestication of Dogs

    • Example: Wild canids were bred into various companion or working dogs.

    • Selection criteria included:

      • Wild and aggressive behavior were selected against.

      • Loyalty to the owner was selected for.

    • Specific breeds were developed for specific jobs:

      • Retrievers for duck hunting,

      • Dachshunds bred to fit in gopher holes.

  • Selective Agriculture

    • Example: Cauliflower, broccoli, kale, and cabbage all come from a common species of plant but were selected for distinct traits.

  • Medicinal Extraction from Plants

    • Examples:

    • Morphine extracted from poppies.

    • Aspirin extracted from willow bark.

    • Fermentation Products: Wine, beer, spirits, cheese, and yogurt are considered forms of "low tech" biotechnology.

Genetic Engineering

  • Definition of Genetic Engineering

    • The altering of an organism’s genome to produce an economically viable product.

  • Examples of Genetic Engineering

    • Inserting Foreign Genes:

    • A gene from another species can be inserted into an organism, enabling it to produce a foreign protein.

    • Example: A gene from salmon inserted into corn to produce an antifreeze protein, allowing the corn to grow in colder temperatures.

    • Another example is inserting a gene that protects plants from destruction by the herbicide glyphosate (found in Roundup).

  • Human Insulin Production

    • Inserting the gene for human insulin into E. coli bacterial cells.

    • Bacteria transcribe and translate the human gene, secreting human insulin protein.

    • This insulin can be purified in large quantities for the treatment of type 1 diabetics.

    • This technology has revolutionized diabetes treatment.

Gene Cloning Process

  1. Excising the Gene of Interest

    • Cutting Out Genes:

      • Use of Restriction Endonucleases (DNA scissors) to excise DNA.

      • These enzymes recognize specific palindromic DNA nucleotide sequences (4 nucleotides long), cutting the DNA at those recognition sites.

      • Example of palindromic sequences:

      • “RACECAR,”

      • “STEP ON NO PETS.”

    • Commercial Availability: Over 200 restriction endonucleases are available for purchase.

      • Bacteria use these enzymes for protection against viral DNA by methylating their own DNA, preventing it from being cut.

  2. Choosing a Restriction Endonuclease

    • Importance of examining the DNA sequence of the target gene including the promoter sequence, start codon, stop codon, and terminator sequence when selecting the appropriate restriction enzyme.

  3. DNA Extraction and Digestion

    • Extract DNA containing the gene of interest and introduce it to the chosen restriction enzyme, e.g., EcoR1, which cuts the sequence GAATTC in the genome.

    • This process leads to hundreds of small DNA fragments.

    • Separation of these fragments using Gel Electrophoresis will help identify the fragment containing the insulin gene, aiding further processing.

Gel Electrophoresis

  • Procedure Overview

    • Setup:

      • Buffer agarose gel designed to allow separation of DNA fragments by size

      • DNA has a negative charge due to phosphate groups, enabling movement towards the positive electrode when an electric current is applied.

    • Sample Preparation:

      • Mix DNA samples with a visible dye to visualize progress and with Ethidium Bromide, an intercalating agent, for UV visualization after separation.

  • Results Interpretation

    • Use of a control lane (DNA ladder) with known DNA fragment lengths to help identify and cut out the band of interest (e.g., insulin gene).

Transformation Process

  • Introduction of Plasmid DNA into Bacteria

    • Bacteria naturally uptake plasmid DNA through Transfection or can be induced to do so through Transformation.

    • Calcium Chloride Induced Competence: Treatments to make bacteria competent for plasmid uptake.

    • Cold temperature (0 degrees C) introduces temporary pores in the membrane, followed by heat shock (42 degrees C) which further facilitates plasmid uptake.

  • Selective Plating

    • To isolate bacteria that have successfully integrated the recombinant plasmid, a nutrient agar plate containing antibiotic is used. Only those that have taken up the plasmid will survive.

Verification and Cloning Steps

  • After transformation, check that antibiotic-resistant bacteria have actually taken up the transgenic plasmid.

  • Gel Electrophoresis is again utilized to confirm the presence of the insulin gene. If present, this indicates a successful cloning process.

  • Final Product: Bacteria reproduce with the plasmid, producing insulin that can be purified for therapeutic use.