Transcript Vectors for Even Larger Genomic DNA Inserts

General Microbiology (Micr300)
Lecture 11
Biotechnology
(Text Chapters: 10.15-10.17; 31.1-31.10)
Definition
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Biotechnology is the use of living organisms
for industrial or commercial application.
It depends heavily on techniques of genetic
engineering.
Much of genetic engineering is based on
molecular cloning, in which a doublestranded DNA fragment from any source is
recombined with a vector and introduced into a
suitable host. Commonly employed cloning
vectors include plasmids and bacteriophages.
Essentials of Molecular Cloning
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A plasmid or virus is used as the cloning
vector to isolate a specific gene or region of a
chromosome by molecular cloning (Figure
10.35).
An in vitro recombination procedure uses
restriction enzymes and DNA ligase to produce
the hybrid DNA molecule. Once introduced into
a suitable host, the cloning vector can control
production of large amounts of the target DNA.
Essentials of Molecular Cloning
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Making a gene library by cloning random
fragments of a genome is called shotgun
cloning, and it is a widely practiced technique
in gene cloning and genomic analyses.
Gene library is defined as a large collection of
DNA fragments cloned (MOLECULAR CLONING)
from a given organism, tissue, organ, or cell
type. It may contain complete genomic
sequences (GENOMIC LIBRARY) or
complementary DNA sequences, the latter
being formed from messenger RNA and lacking
intron sequences.
Plasmids as Cloning Vectors
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Plasmids are useful cloning vectors
(Figure 10.36) because they are easy to
isolate and purify and can multiply to high
copy numbers in bacterial cells.
Antibiotic resistance genes of the plasmid
are used to identify bacterial cells
containing the plasmid (Figure 10.37).
Bacteriophage Lambda as a
Cloning Vector
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Bacteriophages such as lambda have been
modified to make useful cloning vectors
(Figures 10.38, 10.39).
Larger amounts (longer) of foreign DNA can be
cloned with lambda than with many other
plasmids. In addition, the recombinant DNA can
be packaged in vitro for efficient transfer to a
host cell. Plasmid vectors containing the lambda
cos sites are called cosmids, and they can carry
a large fragment of foreign DNA.
Genetic Engineering
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The techniques of genetic engineering
are based on fundamental concepts in
molecular genetics and biochemistry
(Figure 31.1).
Successful genetic engineering depends
not only on being able to carry out
molecular cloning but also on knowledge
of replication, transcription, translation,
and the regulatory aspects that control all
of these processes.
Hosts for Cloning Vectors
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The choice of a cloning host depends on the
final application. In many cases, the host can
be a prokaryote, but in others it is essential that
the host be a eukaryote (Figure 31.2).
Any host must be able to take up DNA, and
there are a variety of techniques by which this
can be accomplished, both natural and artificial.
Figure 31.3 shows a nucleic acid gun for
transfection of certain eukaryotic cells.
Finding the Right Clone
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Special procedures are needed to detect
the foreign gene in the cloning host
(Figure 31.4).
If the gene is expressed, the presence of
the foreign protein itself, as detected
either by its activity or by reaction with
specific antibodies, is evidence that the
gene is present. However, if the gene is
not expressed, its presence can be
detected with a nucleic acid probe.
Specialized Vectors
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Shuttle vectors allow cloned DNA to be moved
between unrelated organisms (e.g., from one bacterium
to another). A shuttle vector is a cloning vector that can
stably replicate in two different organisms.
Many cloned genes are not expressed efficiently in a
new host. Expression vectors have been developed
for both prokaryotic and eukaryotic hosts. These
vectors contain genes that will increase the level of
transcription of the cloned gene and make its
transcription subject to specific regulation (Figure
31.5). Signals to improve the efficiency of translation
may also be present in the expression vector.
Reporter Gene
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Reporter genes are incorporated into
vectors because they encode proteins
that are readily detected. These genes
can be used to signal the presence or
absence of a particular genetic element
or its location. They can also be fused to
other genes or to the promoter of other
genes so that expression can be studied.
Expression of Mammalian Genes
in Bacteria
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It is possible to achieve very high levels
of expression of mammalian genes in
prokaryotes. However, the expressed
gene must be free of introns.
This can be accomplished by using
reverse transcriptase to synthesize cDNA
from the mature mRNA encoding the
protein of interest (Figure 31.8).
Expression of Mammalian Genes
in Bacteria
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One can also use the amino acid
sequence of a protein to design and
synthesize an oligonucleotide probe that
encodes it. This process is in effect
reverse translation and is illustrated in
Figure 31.9.
Fusion proteins are often used to stabilize
or solubilize the cloned protein (Figure
31.10).
Practical Applications:
Production of Insulin
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Production of insulin began the era of
commercial biotechnology.
This was the first human protein made
commercially using engineered bacteria
(Figure 31.11), but many other
hormones and human proteins are now
being produced in this way. In addition,
many recombinant vaccines have been
produced.