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Chapter 9
Biotechnology
and
Recombinant
DNA
SLOs
Compare and contrast biotechnology, recombinant DNA
technology, and genetic engineering.
Identify the roles of a clone and a vector in making
recombined DNA.
Compare selection and mutation.
Define REs, and outline their use to make recombinant DNA.
List some properties of vectors and describe their use.
Outline the steps in PCR and provide an examples of its use.
Describe various different ways of getting DNA into a cell.
Differentiate cDNA from synthetic DNA.
Explain how each of the following are used to locate a clone:
antibiotic-resistance genes, DNA probes, gene products.
Outline advantages of engineering with either E. coli,
Saccharomyces cerevisiae, mammalian cells, or plant cells.
List some advantages of, and problems associated with, the
use of genetic modification techniques.
Terminology and Definitions

Biotechnology: Manipulation (as through genetic
engineering) of living organisms or their components
to produce useful commercial products

Recombinant DNA (rDNA) technology:
Insertion or modification of genes to produce
desired proteins

Clones and cloning:

Cell clones

“to clone a gene” - question: Interest in DNA or in
gene product?
Biotechnology Toolkit

Natural vs. artificial selection

Mutation: Mutagens cause mutations that might result
in a microbe with a desirable trait.
Now: Site-directed mutagenesis

Restriction Enzymes (RE): Molecular scissors

Cut specific sequences of DNA

Destroy bacteriophage DNA in bacterial cells

Methylases protect own DNA by methylating cytosines

Bunt ends vs. sticky ends
Review Microbiology Animations with Quizzes in
Mastering Microbiology
Site of cleavage
Restriction Enzymes = REs
(= Restriction Endonucleases)
EcoRI
Recognition
Fig 8-25
sequence
is
always a
palindrome
Review Table 9.1
Origin and Naming of Restriction Enzymes
Role of Restriction Enzymes in Making
Recombinant DNA Molecules
Fig 9.2
Vectors



Also known as cloning vectors.
Must be
 Small and easy to manipulate. ________ & _________
serve as vectors.
 self-replicating
 large quantities
When they carry “insert”:
= Recombinant DNA molecules

Introduce foreign DNA
(desired gene) into host cells

Shuttle vectors can exist
in several different species.
... One of most commonly used vectors:
Polymerase Chain Reaction (PCR)

Makes multiple copies of a piece of DNA
enzymatically

Used to

Clone DNA for recombination

Amplify DNA to detectable levels

Sequence DNA

Diagnose genetic disease

Detect pathogens
Fig 9.4
Review Microbiology Animations with Quizzes in
Mastering Microbiology
Techniques of Genetic Modification
Inserting Foreign DNA into
Cells

DNA can be inserted into a
cell by

Transformation

Protoplast fusion

Electroporation

Microinjection
Fig 9.7
Blue and White Screening Method
for Selecting a Clone (or Recombinant DNA Molecule)
Direct selection of engineered vector via antibioticresistance markers (ampR) on plasmid vectors.
Vector also contains-galactosidase gene for bluewhite screening
Desired gene is inserted into the -galactosidase gene site
 gene inactivated
Three possible outcomes:
1.
Bacteria lack vector  _______________
2.
Bacterial clones contain vector without the new gene
 colony type? _______________
Bacterial clones contain recombinant vector 
resistant to Ampicillin and unable to hydrolyze X-gal
 colony type? _______________
3.
Possible Method to
detect
recombinant
bacteria:
Blue–White
Screening
1) Plasmid cloning
Fig 9.11
2) Selecting Recombinant Bacteria
Which type of
colonies do you
want?
a)White
b)Blue
c)I don’t want
any
Making a Gene Product
E. coli: prokaryotic workhorse of biotech. Easily grown
and genomics well understood. Disadvantage:
Cells must be lysed to get product  release of ______
Yeast: Saccharomyces cerevisiae is eukaryotic
workhorse of biotechnology. Advantage: Continuous
secretion of gene product.
Mammalian cells: May express eukaryotic genes easily.
Disadvantage: Harder to grow.
Plant cells: Easy to grow. May express eukaryotic genes
easily.
Some Applications of DNA Technology
Forensic Microbiology & Diagnostics: PCR and
DNA probes can be used to quickly identify a
pathogen in body tissue or food.
Therapeutic Applications:
1. Insulin production
See Table 9.2
2.
Subunit vaccines
3.
DNA vaccines
4.
Gene therapy to replace defective or missing
genes
5.
Pharmaceutical applications

Hormone and Antibiotics production
Safety Issues and Ethics of Using rDNA
Strict safety standards avoid accidental release of
genetically modified microorganisms.
Some microbes used in cloning have been altered so that
they cannot survive outside the laboratory.
Microorganisms intended for use in the environment may
be modified to contain suicide genes  organisms do
not persist in the environment.
Safety and ethical concerns beyond microbiology: Who will
have access to an individual's genetic information? Are
genetically modified crops safe for release to environment?
A Typical Genetic Modification Procedure
Foundation Figure
Fig 9.1