Lecture Notes: Enzyme Science and Technology

Overview

• Continuing discussion on enzyme science and technology.
• Focus on enzyme properties and DNA delivery into host cells.

Previous Modules Recap

History and Structure of Enzymes

• Development of enzymology.
• Enzyme structure: primary, secondary, tertiary, and quaternary.
• Techniques for determining enzyme structures.

Gene Isolation and Cloning

• Methods to isolate gene fragments from genomes.
• Cloning fragments into vectors.
• Delivery of vectors into suitable hosts.

Current Module: DNA Delivery into Host Cells

• Strategies for DNA delivery into host cells.
• Scheme for enzyme production.
  • Gene isolation using genomic or cDNA libraries or PCR.
  • Digestion and ligation to create recombinant clones.

DNA Delivery in Host Cells

Host Cell Surface Chemistry

• DNA is negatively charged; interacts with host cell surfaces.
• Importance of cell surface charge:
  • Positive charge attracts DNA.
  • Negative charge repels DNA.
• Physical barriers like cell walls affect DNA entry.

Competent Cells and Transformation

• Wild-type cells are not naturally competent.
• Competent cells: cells treated to change surface chemistry to uptake DNA.
• Transformation: process of DNA uptake by a host cell.
  • Example: antibiotic resistance transfer among bacteria.

Mechanism of Transformation

• DNA from donor binds to competent recipient cells.
• DNA enters cells and integrates into chromosomal DNA via homologous recombination.
• Transformation common among closely related species.

Laboratory Transformation Methods

• Chemical methods (using agents like calcium chloride).
• Electroporation (using electrical pulses to create membrane pores).

Preparing Competent Cells

Chemical Transformation

• Example: E. coli transformation using calcium chloride.
• Process involves growing cells to log phase, incubation with divalent cations, and heat shock treatment.
• Sensitive handling due to fragile state post-treatment.

Electroporation

• Utilizes electrical pulses for DNA entry.
• Does not depend on surface chemistry; requires specialized equipment.

Factors Affecting Transformation Efficiency

• Plasmid size and form (supercoiled vs. open/closed circular).
• Genotype of host cells.
• Growth phase of cells (log phase preferred).
• Transformation method and DNA quality.

Yeast Transformation Methods

Lithium Acetate Method

• Uses lithium acetate, PEG, and single-stranded DNA to facilitate transformation.

Spheroplast Method

• Partial cell wall removal with zymolase.
• Incubation with carrier DNA and PEG for DNA uptake.

Conclusion

• Discussed chemical and electroporation methods for DNA delivery into bacterial and yeast cells.
• Next lecture will cover DNA delivery into mammalian cells.