4E- recombinant and plasmid DNA

Introduction to Insulin Production via Bacteria

  • Insulin production has transitioned from using pig and cow pancreases to utilizing bacteria for several reasons:
    • Ethical concerns surrounding animal slaughter for medical purposes.
    • The cleanliness and purity of the synthesized insulin produced by transformed bacteria.

Key Concepts in Genetic Engineering

  • Transforming Bacteria:

    • The process of introducing new DNA into bacteria.
    • The goal is often for the bacteria to produce a specific protein, such as insulin (a protein).

  • Types of DNA Endings:

    • Sticky Ends:
    • Considered more specific for genetic engineering.
    • They have overhanging bases that allow for matching with complementary sequences on other DNA strands.
    • This specificity helps ensure correct genetic joins.
    • Blunt Ends:
    • Do not have overhanging bases, allowing them to attach to any compatible end without specificity.
    • While convenient for some applications, they introduce uncertainty in ensuring correct attachments.

Plasmids

  • Definition of a Plasmid:
    • A plasmid is a small, circular piece of DNA found in bacteria.
    • It is distinct from chromosomal DNA, as it contains genes for survival traits rather than essential life-supporting genes.
    • Function: Plasmids can be exchanged between bacteria, providing a method for sharing beneficial traits.
  • Recombinant Plasmid:
    • A plasmid that has been modified to include external DNA (often containing a gene of interest).

Process of Creating a Recombinant Plasmid

  • Steps Involved:
    1. Selection of Restriction Endonuclease:
    • A specific enzyme that makes cuts in DNA to create sticky ends.
    • The selected endonuclease must cut the DNA near the gene of interest.
    1. Cloning Process - PCR:
    • Use Polymerase Chain Reaction (PCR) to make multiple copies of the target gene.
    1. Cutting the Plasmid:
    • The same restriction endonuclease is used to cut the plasmid, leaving sticky ends for joining.
    1. Joining the DNA Samples:
    • Mix the cut plasmid and the amplified gene with DNA ligase to form phosphodiester bonds.
    • This produces a recombinant plasmid.
    • There is a potential problem during this step: DNA ligase may reattach the plasmid without incorporating the gene.
    1. Introduction of Recombinant Plasmid to Bacteria:
    • Bacteria need to be made competent (able to take in new DNA).
    • Shock methods (like heat shock) are typically used to induce competence.
    1. Selection of Transformed Bacteria:
    • Bacteria that uptake the plasmid may or may not carry the desired gene.
    • Additional characteristics (like antibiotic resistance or the ability to digest lactose) help in identifying successful transformations.

Challenges and Considerations

  • Two major potential issues:

    1. The ligation step could fail to incorporate the desired gene, leaving the plasmid intact but inactive.
    2. The bacteria may not uptake the plasmid at all, resulting in unsuccessful transformations.

  • Identification Process:

    • In addition to the gene of interest, a selectable marker is introduced (like antibiotic resistance or lactase).
    • After transformation, bacteria are placed on media lacking certain nutrients or containing antibiotics; only those with the recombinant plasmid survive.
    • Fluorescent markers can also be used to visually identify successful transformations under UV light.

Summary of Genetic Engineering Process

  1. Choose a gene and an appropriate restriction endonuclease.
  2. Cut the gene and plasmid with the selected endonuclease to create sticky ends.
  3. Amplify the gene via PCR to produce more copies.
  4. Join the amplified gene to the plasmid using DNA ligase, creating a recombinant plasmid.
  5. Make bacteria competent for uptake of the plasmid.
  6. Select for transformed bacteria using media that only supports survival of bacteria with the recombinant plasmid.

Ethical Considerations in Genetic Engineering

  • Discussions around GMOs address various topics:
    • Ethical implications of creating modified organisms.
    • The profitability of biotechnology, with concerns over monopolies in pharmaceutical and agricultural industries.
    • Consideration of societal benefits vs. potential risks involved in genetic modifications.
  • Future chapters may explore in depth the ethical implications of GMOs, requiring grounding in various ethical perspectives, such as:
    • Virtues-based approaches to ethics.
    • The principle of non-maleficence (avoiding harm).
    • Justice in distribution of biotechnological benefits.
    • Economic implications regarding who profits from biotechnological advances.