Genetic Engineering Learning Outcomes PDF

Learning Outcomes ! Outline the fundamental steps in a genetic engineering procedure and give examples of genetically engineered products ! Describe the mechanism of action and the use of restriction enzymes in biotechnology research and recombinant protein production ! Discuss techniq...

Learning Outcomes ! Outline the fundamental steps in a genetic engineering procedure and give examples of genetically engineered 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 of a bacterial transformation and various selection processes for identifying transformants ! Differentiate transformation, transfection, and transduction ! Discuss the considerations for scaling up the production of transformed or transfected cells, the general cell culture protocol for scale-up, and the importance of complying with standard manufacturing procedures ! Explain the usefulness of plasmid preparations, how they are performed, and how the concentration and purity of plasmid samples can be determined  8.1 An Overview of Genetic Engineering The goal of genetic engineering is to produce organisms with new, improved characteristics. Genetic Engineering to Produce a Protein Product 1. Recombinant DNA Technology ! The genetic code (DNA) for the desired characteristic or protein is identified and isolated from a donor cell, and pasted into a vector, producing a recombinant DNA [rDNA] plasmid, that can carry the desired DNA code into a recipient cell. 2. Transformation ! Genetically engineered cells are produced when the vector carrying the gene of interest is transferred into new host cells. If the cells express the new DNA, transcribing and translating it into a new (recombinant) protein, the cells are “transformed.” 3. Cloning ! The transformed cells, producing their recombinant protein, are grown in culture (cloning). First, they are grown on a small scale in Petri plates and small broth cultures up to 10L (fermentation) and scaled up to larger volumes, up to 30,000L (manufacturing). 4. Purification ! Finally, the recombinant protein product, being produced in manufacturing, must be isolated and purified from the cells and other proteins in the cell culture. Before purified recombinant protein product is sent to market, it is tested for purity and may have to go through governmental approvals. Isolating Genetic Information Genomic DNA Extraction Kits This is an autoradiogram, a visual record of a gel analysis created on film by the reaction of small amounts of radioactivity present in each sample of DNA. Each band represents thousands of identical fragments of amplified DNA produced by PCR. Genomic (chromosomal) DNA is extracted from cells for this type of lab work. Probing DNA for Genes of Interest Probing DNA. A probe is used to identify certain DNA sequences that are hidden among the billions of nucleotides in a genome. Using Polymerase Chain Reaction (PCR) to Locate Genes of Interest A thermal cycler is used to make millions of copies of DNA fragments in just a few hours using PCR reagents. Underneath the cover is a heat block that holds 96 sample tubes that are cycled through different temperatures. 8.2 Using Recombinant DNA (rDNA) for Transformation Making rDNA Big Picture View of Genetic Engineering DNA Fingerprint In these samples, a mutation in a gene is recognized by a difference in banding patterns. In DNA fingerprinting, RFLP analysis gives unique banding patterns because each person’s DNA code is unique.  8.3 Transforming Cells Uptake and expression of foreign DNA by a cell General Steps of a Transformation Grow the host cells in broth culture. Place cell culture on ice, make them competent (ready to take up DNA) with CaCl2 or MgCl2. Add rDNA plasmid sample to the competent cells. Heat shock the cells by rapidly moving them from ice to a water bath (37°C or 42°C) for 20-90 seconds (depending on the strain of cells) and then quickly back on ice. Add a nutrient broth for cell recovery and gene expression at some optimum temperature (37°C for E. coli). Plate out the cells on some kind of selection media/agar that shows that the cells are producing the new protein.  Steps in a Typical Transformation Steps in a Typical Transformation 8.4 After Transformation - Manufacturing At a biotechnology company, the goal is to produce enough of a product to sell and make a profit. The profit is reinvested into the company for more R&D. The Scale-Up Process To produce enough product, transformed cells must be scaled up into progressively larger volumes of broth culture. Here, cell cultures in four 2-L spinner flasks are suspended and aerated. The amount of oxygen the cells are receiving is the most critical factor in growing the culture at a maximum rate. From Scale-Up to Fermentation to Manufacturing Bioreactor/Fermentation Tank A technician prepares a bioreactor/fermentation tank for inoculation with cell culture. It must be cleaned thoroughly and sterilized before any culture is added.  Using Assays during Scale-Up During scale-up assays measure protein concentration and protein activity as well as other variables. 8.5 Fermentation, Manufacturing, and GMP Fermentation is the process by which cells utilize glucose for cellular energy under anaerobic conditions. alcoholic fermentation: glucose ➞ carbon dioxide + ethanol lactic acid fermentation: glucose ➞ lactic acid  To ensure healthy cells that produce protein at a maximum rate, cultures are grown to exponential growth as measured as an OD600 of about 0.6au. Before the growth rates decrease below A technician cleans a 5-L fermenter the level of exponential growth, the following current cGMP and validates sample is harvested or used to see a it for clean-in-place effectiveness. larger vessel. 8.6 Retrieving Plasmids after Transformation It is often necessary to extract the transforming plasmid from transformed cells.  Types of Plasmid “Preparation” Miniprep Midiprep Maxiprep Reasons for Performing a Prep Gigapreps Ensures that transformation has actually occurred Collect more plasmids for future transformations Testing for the Presence of DNA Indicators (like dotMETRIC) Ethidium bromide (EtBr) dot test UV spectrophotometer  DNA Concentration Equation 50 μg/mL = X μg/mL _________ ________________ 1 au at 260 nm the au of sample at 260 nm  DNA Purity Equation absorbance (au) at 260 nm ________________ = the purity value absorbance (au) at 280 nm