Genetic Engineering Overview

Learning Outcomes

• Outline the fundamental steps in a genetic engineering procedure and describe examples of such products.
• Describe the mechanism of action and the use of restriction enzymes in biotechnology research and recombinant protein production.
• Discuss techniques used to probe DNA for specific genes of interest.
• Explain the steps involved in bacterial transformation and various selection processes used to identify transformed cells.
• Differentiate transformation, transfection, and transduction.
• Discuss considerations for scaling up the production of transformed or transfected cells, including the general cell culture protocol for scale-up, the importance of adhering to standard manufacturing procedures, and the usefulness of plasmid preparations.
• Explain how plasmid preparations are performed, and how to determine the concentration and purity of plasmid samples.

Genetic Engineering Overview

• The goal of genetic engineering is to create organisms with improved characteristics.
• Steps in genetic engineering:
  • Step 1: Identification and insertion of the gene of interest.
  • Step 2: Recombinant DNA (rDNA) vector introduction into host cells.
  • Step 3a & 3b: Cell division/reproduction on small and large scale.
  • Step 4: Purification of the recombinant protein.

Genetic Engineering to Produce a Protein Product

• Recombinant DNA Technology: Isolate the desired DNA from a donor cell and insert it into a vector to create recombinant DNA.
• Transformation: Transfer the recombinant DNA into host cells to produce a protein.
• Cloning: Grow the transformed cells in culture to produce more cells and proteins.
• Purification: Isolate the desired protein from the cells and other proteins.

Isolating Genetic Information

• Genomic DNA Extraction Kits are used to isolate genomic DNA.
• Autoradiograms are visual records of gel analysis, showing the presence of DNA fragments.

Probing DNA for Genes of Interest

• Probes are used to identify specific DNA sequences.
• Probes are labeled with radioactivity to detect specific gene sequences in genome.

Using Polymerase Chain Reaction (PCR) to Locate Genes of Interest

• Polymerase Chain Reaction (PCR) is used to amplify specific DNA sequences.
• A thermal cycler is used for PCR, cycling through different temperatures to amplify DNA.

Using Recombinant DNA (rDNA) for Transformation

• Restriction enzymes are used to cut DNA at specific sites, creating sticky ends.
• DNA ligase joins DNA fragments with matching sticky ends, creating recombinant DNA.

Big Picture View of Genetic Engineering

• The same restriction enzyme cuts both pieces of DNA, resulting in matching sticky ends.
• The spliced section is then inserted into the plasmid vector.
• The enzyme DNA ligase seals the matching sticky ends.
• rDNA is inserted into a bacterial cell.
• Transformed cell expresses the newly acquired DNA by producing mRNA and the protein encoded by it.

DNA Fingerprint

• DNA samples, with a mutation in a gene, can be identified by differences in banding patterns.
• RFLP (Restriction Fragment Length Polymorphism) analysis yields unique banding patterns for each person's DNA.

Transforming Cells

• Steps in transforming cells:
  • Grow host cells in a broth culture.
  • Make cells competent to take up DNA using CaCl2 or MgCl2.
  • Add the rDNA plasmid to the competent cells.
  • Heat shock the cells and then return them to ice.
  • Add a nutrient broth to recover and express the new gene.
  • Plate out the cells on selection media.

After Transformation - Manufacturing

• In biotechnology companies, the aim is to produce a sufficient quantity of a product for sale and reinvest profits in R&D.
• The scale-up process involves progressively larger volumes of broth cultures (from small flasks to very large tanks.)
• Adequate oxygenation is crucial for maximum cell growth during scale-up.

From Scale-Up to Fermentation to Manufacturing

• Transformed cells are cultured from small volumes to large volumes of broth in different types of vessels.

Bioreactor/Fermentation Tank

• The bioreactor or fermentation tank must be thoroughly cleaned and sterilized before use.

Using Assays During Scale-Up

• Assays are used to measure protein concentration and protein activity during the large scale production of transformed cells.

Fermentation, Manufacturing, and GMP

• Fermentation is an anaerobic process where cells utilize glucose for energy.
• Types of fermentation include alcoholic fermentation (produces ethanol) and lactic acid fermentation (produces lactic acid).

Retrieving Plasmids after Transformation

• Plasmids are extracted from transformed cells to be used in further transformations.
• Steps include cell lysis, spin, isopropanol, TE buffer and isopropanol again to recover the plasmids.

Types of Plasmid "Preparation"

• Different types of plasmid preparations exist (miniprep, midiprep, maxiprep, gigaprep) suited for various purposes and amounts of DNA.

Testing for the Presence of DNA

• Indicators and techniques to test for DNA presence (dotMETRIC, ethidium bromide, UV spectrophotometer) are utilized to determine plasmid amount and purity.

DNA Concentration Equation

• Formula to calculate the concentration of extracted DNA from absorbance measurements at 260 nm (50 µg/mL = X µg/mL / 1 AU at 260 nm).

DNA Purity Equation

• Formula to calculate the purity of extracted DNA from absorbance measurements at 260 nm and 280 nm. Calculated as: (absorbance at 260 nm)/(absorbance at 280 nm) gives the purity value).

This quiz explores key concepts in genetic engineering, including procedures, the role of restriction enzymes, and methods for DNA probing. It covers essential techniques such as bacterial transformation and the considerations for scaling up production. Test your understanding of plasmid preparations and the differences between transformation, transfection, and transduction.