Genetic Engineering Overview

Learning Outcomes

• Genetic engineering aims to create organisms with improved traits.
• Steps in genetic engineering include outlining fundamental steps, providing examples of genetically engineered products, describing restriction enzyme mechanisms and uses in biotechnology, discussing DNA probing techniques, explaining bacterial transformation and selection processes, differentiating transformation, transfection and transduction, discussing considerations for large-scale production, explaining plasmid preparation, and determining plasmid concentration and purity. This includes understanding the process of isolating genetic information and using techniques like PCR and DNA probes.

Overview of Genetic Engineering

• The goal of genetic engineering is to produce organisms with new, improved characteristics.
• This involves several steps:
  • Inserting the gene of interest into a plasmid (Step 1 and 2)
  • Culturing the recombinant cells (Step 3a and 3b)
  • Purifying the desired protein (Step 4)
  • Steps commonly follow the pattern: insertion, culture, purification, and, potentially, testing and market release.

Genetic Engineering to Produce a Protein Product

• Recombinant DNA Technology: The desired genetic code (DNA) for a characteristic or protein is isolated from a donor cell and inserted into a vector, creating a recombinant DNA plasmid. The plasmid carries the desired DNA code to a recipient cell.
• Transformation: Genetically engineered cells are produced when the vector with the gene of interest is transferred into a new host cell. If the cells express the new DNA, by transcribing and translating it into a protein, they are transformed, resulting in expression of the new gene.
• Cloning: Transformed cells producing their recombinant protein are cultured (cloned). This is done first on a small scale (Petri plates, small broth cultures) before scaling up to larger volumes (10 L, 30,000 L), and potentially even larger volumes, for manufacturing.
• Purification: The recombinant protein product is isolated and purified from the cells and other proteins in the culture. Before being sold, the product is tested for purity and may go through governmental approval. This includes understanding the entire production process, from initial transformation to final purification and approval.

Isolating Genetic Information

• Genomic DNA Extraction Kits are used to isolate genomic DNA from cells, for instance, for preparing samples for gel analysis.
• Autoradiograms visualize the results of gel analyses by showing the reaction with radioactive small amounts of DNA. Each band represents thousands of identical amplified DNA fragments that are analyzed.

Probing DNA

• A probe is used to identify specific DNA sequences within a genome.
• The probe is a piece of DNA with a radioactive label, complementary to the gene of interest. The probe locates a specific gene of interest.
• Probes are washed over the gel.
• The probe binds to its complementary sequence in the sample and emits radiation, which is visible on X-ray film. This enables the identification of specific sequences within the sample.

Using Polymerase Chain Reaction (PCR)

• A thermal cycler is used to make millions of copies of DNA fragments within a few hours using PCR reagents.
• The thermal cycler cycles the sample through different temperatures for specific DNA reactions, often used to amplify a specific gene region.

Using Recombinant DNA for Transformation

• Restriction enzymes are used to cut open a plasmid DNA molecule.
• A section of DNA containing the gene of interest is spliced out of a DNA molecule from a second source, using the same restriction enzyme.
• DNA ligase seals the matching sticky ends, creating a new molecule with DNA from both sources (recombinant DNA). The process carefully links matching DNA segments.

Big Picture View of Genetic Engineering

• A plasmid vector is opened by a restriction enzyme.
• The same restriction enzyme cuts both pieces of DNA so they have matching sticky ends, facilitating the assembly of rDNA.
• The spliced section is pasted into the plasmid vector.
• The enzyme DNA ligase seals the matching sticky ends, placing the rDNA into a bacteria cell.
• The transformed cell then expresses the newly acquired DNA by producing mRNA and the protein encoded from it.

DNA Fingerprinting

• In DNA fingerprinting, mutations in a gene can be identified based on differences in banding patterns.
• The RFLP (Restriction Fragment Length Polymorphism) analysis produces the unique banding patterns, often used to differentiate individual DNA samples.

Transforming Cells

• Host cells are grown in a broth culture.
• The culture is placed on ice to make cells competent to absorb DNA using chemicals such as CaCl₂ or MgCl₂, which prepares the cells to receive the foreign DNA.
• The rDNA plasmids are then added to the competent cells.
• Cells are heat-shocked (moved to 37°C or 42°C), then returned to ice. This process helps the DNA enter the cells.
• Nutrient broth is added for recovery and protein expression.
• Cells are plated on selection media to identify cells producing the new protein. Selection media ensures only transformed (successful) cells survive and reproduce.

After Transformation - Manufacturing

• The goal is to produce enough of the product to generate profit, which is then reinvested in R&D to improve the entire process and develop new technologies.
• The scale-up process involves progressively increasing the culture volumes (from small flasks to larger tanks and eventually 3,000 - 10,000 L tanks), to meet the demands of production.
• Cell cultures in spinner flasks are often used and oxygen supply is critical for proper growth rate. This ensures that cells grow at a high rate, efficiently producing the protein of interest.

From Scale-Up to Fermentation to Manufacturing

• Transformed cells are transferred from selection agar to broth culture.
• The scale of culture increases (from 1 liter to 2 liter, then 100 liter and beyond). In this process, the controlled culture volumes are a key part of the process to ensure high yields and production rate.

Bioreactor/Fermentation Tank

• Bioreactors/fermentation tanks are cleaned and sterilized before cell culture is inoculated. Preparation and sterilization ensure proper environmental condition for cell growth and prevent contamination.

Using Assays During Scale-Up

• Assays are used to determine protein concentration and activity during scale-up. Assays provide consistent and monitored data on the process.

Fermentation, Manufacturing, and GMP

• Fermentation utilizes glucose for energy under anaerobic conditions by cells.
• This process has two common types: alcoholic fermentation and lactic acid fermentation. Techniques must follow industry standards.

Retrieving Plasmids after Transformation

• Plasmids are extracted from transformed cells.
• Cells are lysed (broken open) and the plasmids separated.
• Plasmid concentration is measured using a spectrophotometer. This is an essential step in determining plasmid success.

Types of Plasmid "Preparation"

• Different types of plasmid preparations exist (Mini, Midi, Maxi, Giga) with varying scales. This is important since the scale must match the needs of the project.

Testing for the Presence of DNA

• Indicators, such as dotMETRIC, are used to detect the presence of DNA.
• Ethidium Bromide (EtBr) dot test is another indicator.
• UV spectrophotometer quantifies DNA concentration and purity.

DNA Concentration Equation

• The equation to determine DNA concentration in a sample is: 50 µg/mL = X µg/mL / 1 au at 260 nm.

DNA Purity Equation

• DNA purity is measured by: absorbance (au) at 260 nm / absorbance (au) at 280 nm. This ratio helps determine the purity of the isolated DNA.

Explore the fundamental concepts and methods of genetic engineering through this quiz. Learn about the steps involved, techniques like DNA probing, and applications in biotechnology. This quiz is ideal for students looking to understand the intricacies of producing genetically modified organisms.