The Production of aRecombinant Biotechnology ProductChapter 8

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 can be determined with a UV spectrophotometer

8.1 An Overview of Genetic Engineering

Goal of genetic engineering is to produce organisms with new, improved characteristics.Isolating Genetic Information

This is an autoradiogram, which is a visual record of gel analysis created on film by the reaction of small amounts of radioactivity present in each sample of DNA. The samples have been processed using the polymerase chain reaction (PCR), a technique in which small sections of the DNA are amplified from very small starting amounts. Each band on the autoradiogram represents thousands of identical fragments of amplified DNA. Genomic (chromosomal) DNA must be extracted from cells for this type of work. DNA may be isolated from cells using a genomic DNA purfication kit.

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 heatblock that holds 96 sample tubes that are cycled through different temperatures.

Vocabulary

• Lysozyme – an enzyme that degrades bacterial cell walls by decomposing the carbohydrate peptidoglycan

• Supernatant – the (usually) clear liquid left behind after a precipitation has been spun down to the bottom of a vessel by centrifugation

• Probe – a DNA or RNA molecule that is complementary to the DNA sequence being investigated, often bound to some kind of “reporter” molecule, used when looking for a gene or nucleic acid sequence

• Hybridization – the binding of complementary nucleic acids• Autoradiogram – the image on an x-ray film that results from exposure to

radioactive material• Southern blotting – a process in which DNA fragments on a gel are transferred

to a positively charged membrane (a blot) to be probed by labeled RNA or cDNA fragments

• Blot – a membrane that has proteins, DNA, or RNA bound to it• cDNA – abbreviation for “copy DNA.” cDNA is DNA that has been synthesized from

mRNA• Primers – small strands of DNA used as starting points for DNA synthesis or

replication• Thermal cycler – an instrument used to complete PCR reactions; automatically

cycles through different temperatures

8.1 Review Questions

1. What is the name of the genetically engineered rennin molecule? Why is it desirable to produce genetically engineered rennin instead of harvesting rennin from nature?

2. What is the name of the process that occurs when complementary pieces of DNA or RNA locate and bind to each other? How is that technology used in the laboratory?

3. What is a Southern Blot, and how is it used?

4. What does a thermal cycler do?

8.2 Transforming Cells

Uptake and expression of foreign DNA by a cell

Making Recombinant DNA

Applications of RFLP Analysis

A DNA fingerprint is the unique pattern that results from the DNA analysis of an individual

Performing a Transformation

Transformations do occur in nature; this is how some species of bacteria quickly acquire new traits

Steps in a Typical Transformation

Vocabulary

• Transformation – the uptake and expression of foreign DNA by a cell• Transduction – the use of viruses to transform or genetically engineer cells• Endonucleases – enzymes that cut RNA or DNA at specific sites; restriction enzymes

are endonucleases that cut DNA• Sticky cells – restriction fragments in which one end of the double stranded DNA is

longer than the other; necessary for the formation of recombinant DNA• Restriction enzyme mapping – determining the order of restriction sites of enzymes

in relation to each other• Competent/competency – the ability of cells to take up DNA• Selection – the process of screening potential clones for the expression of a particular

gene, for example, the expression of a resistance gene (such as resistance to ampicillin) in transformed cells

• Transformation efficiency – a measure of how well cells are transformed to a new phenotype

• Recovery period – the period following transformation where cells are given nutrients and allowed to repair their membranes and express the “selection gene(s)”

• Beta-galactosidase gene – a gene that produces beta-galactosidase, an enzyme that converts the carbohydrate X-gal into a blue product

• Green fluorescent protein – a protein found in certain species of jellyfish that glows green when excited by certain wavelengths of light (fluorescence)

• Scale-up – the process of increasing the size or volume of the production of a particular product

8.2 Review Questions

1. What is the name of the process in which bacteria receive and express a) recombinant plasmid DNA; b) recombinant viral DNA? What is the name of the process in which mammalian cells receive and express rDNA?

2. Which two types of enzymes are needed to produce an rDNA molecule?

3. What is the name for the differences in gel banding patterns in DNA samples that result from a restriction enzyme’s activity?

4. Which two techniques are used to increase transformation efficiency?

8.3 After Transformation

The Scale-Up Process

Cell cultures in four 2-L spinner flasks. Each flask has a spinner apparatus (propeller blade) inside to keep the cells 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

Using Assays during Scale-Up

Assays measure protein concentration and activity

Vocabulary

• Spinner flasks – a type of flask commonly used for scale-up in which there is a spinner apparatus (propeller blade) inside to keep cells suspended and aerated

• Fermenters – automated containers used for fermentation, when cultures are easily monitored and controlled

8.3 Review Questions

1. What is a spinner flask and where is it used?

2. What type of environmental conditions must be monitored in cultures as they are scaled up?

3. How are ELISAs utilized during manufacturing?

4. How is a QC department involved in the manufacturing process?

8.4 Fermentation, Manufacturing, and GMP

Fermentation is the process by which cells utilize glucose under anaerobic conditions.

To ensure healthy cells that produce protein at a maximum rate, cultures are grown to exponential growth. Before the growth rates decrease below the level of exponential growth, the sample is harvested or used to see a larger vessel.

A technician cleans a 5-L fermenter following current cGMP and validates it for clean-in-place effectiveness.

Vocabulary

• Alcoholic fermentation – a process by which certain yeast and bacteria cells convert glucose to carbon dioxide and ethanol under anaerobic (low or no oxygen) conditions

• Lactic acid fermentation – a process by which certain bacteria cells convert glucose to lactic acid under anaerobic (low or no oxygen) conditions

• Seed – the initial colony of a culture that is used as starter for a larger volume of culture

• Exponential growth – the growth rate that bacteria maintain when they double in population size every cycle

• Lag phase – the initial period of growth for cells in culture after inoculation

• Stationary phase – the latter period of a culture in which growth is limited due to the depletion of nutrients

• cGMP – abbreviation for current good manufacturing practices

8.4 Review Questions

1. Distinguish between the processes of alcoholic fermentation and lactic-acid fermentation. Give an example of a product made by each process.

2. Manufacturing teams want to keep cell cultures in exponential growth. What is exponential growth?

3. Place these events in order, from early in the product pipeline to late in the pipeline.Transformation ManufacturingFermentation R&DAssay Development QCScale-Up

4. Which federal agency is responsible for setting cGMP guidelines?

8.5 Retrieving Plasmids after Transformation

It is often necessary to extract the transforming plasmid from transformed cells.

Reasons for Performing a Prep

Ensures that transformation has actually occurred

Collect more plasmids for future transformationsTesting for the Presence of DNA

Ethidium bromide (EtBr) dot test

UV spectrophotometer

Vocabulary

• Preparation – the process of extracting plasmids from cells

• Miniprep – a small DNA preparation yielding approximately 20 g/500 L of plasmid DNA

• Midiprep – a DNA preparation yielding approximately 800 g/mL of plasmid DNA

• Maxiprep – a DNA preparation yielding approximately 1 mg/mL or more of plasmid DNA

8.5 Review Questions

1. What is the name of the procedure in which plasmids are extracted from cells?

2. How is plasmid DNA precipitated in the final steps of a plasmid prep?

3. Once plasmid is extracted from a cell, how can a technician know that it is the “correct” plasmid?

4. If a DNA sample gives a 260-nm reading of 0.8 au and a 280-nm reading of 0.5 au, what are its concentration and purity? Is this purity acceptable?

Questions and Comments?