Download The Production of a

The Production of a
Recombinant Biotechnology
Product
Chapter 8
Learning Outcomes
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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
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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
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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
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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
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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
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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
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Lactic acid fermentation – a process by which certain bacteria cells convert glucose
to lactic acid under anaerobic (low or no oxygen) conditions
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Seed – the initial colony of a culture that is used as starter for a larger volume of
culture
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Exponential growth – the growth rate that bacteria maintain when they double in
population size every cycle
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Lag phase – the initial period of growth for cells in culture after inoculation
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Stationary phase – the latter period of a culture in which growth is limited due to the
depletion of nutrients
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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
late in the pipeline.
Transformation
Fermentation
Assay Development
Scale-Up
4.
order, from early in the product pipeline to
Manufacturing
R&D
QC
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 transformations
Testing for the Presence of DNA
Ethidium bromide (EtBr) dot test
UV spectrophotometer
Vocabulary
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Preparation – the process of extracting plasmids from cells
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Miniprep – a small DNA preparation yielding approximately 20 mg/500 mL of plasmid DNA
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Midiprep – a DNA preparation yielding approximately 800 mg/mL of plasmid DNA
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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?