Chapter 31

  Review of Principles Underlying Genetic Engineering
Biotechnology
Biotechnology is the use of living organisms to carry out chemical processes for industrial or commercial application.

genetic engineering is based on molecular cloning
a double-stranded DNA fragment from any source is recombined with a vector and introduced into a suitable host.
cloning vectors include plasmids and bacteriophages.


The choice of a cloning host depends on the final application.
In many cases, the host can be a prokaryote, BAC
In others it is essential that the host be a eukaryote. YAC


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.


Special procedures are needed to detect the foreign gene in the cloning


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.

If the gene is not expressed, its presence can be detected with a nucleic acid probe.

Shuttle vectors allow cloned DNA to be moved between unrelated organisms.
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.

Expression vectors contain genes that will increase the level of transcription of the cloned gene and make its transcription subject to specific regulation.
Signals to improve the efficiency of translation may also be present in the expression vector.

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.

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
Reverse Translation
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.


Fusion proteins are often used to stabilize or solubilize the cloned protein.


The first human protein made commercially using engineered bacteria was human insulin, but many other hormones and human proteins are now being produced. In addition, many recombinant vaccines have been produced.