Biomedical Engineering Reference
In-Depth Information
PCR. Another approach is DNA shuffling, which requires genes from homologous proteins.
Segments from the genes are recombined randomly to form chimeric genes. Proteins are typi-
cally selected for improved stability, binding strength, catalytic activity, or solubility. In some
cases, the screen is for new activities based on ability to bind other molecules not normally
bound by the native protein. These techniques combined with improved biochemical
methods are leading to a better understanding of the relation of protein structure to function.
Also, these techniques support the extension of protein catalysis to unusual environments,
such as in organic media rather than in aqueous solutions.
14.11. SUMMARY
A cell's genotype represents the cell's genetic potential, whereas its phenotype represents the
expression of a culture's potential. The genotype of a cell can be altered by mutations. Exam-
ples of mutations are point mutations, deletions , and additions . Additions are usually the result
of insertion sequences that “jump” from one position to another.
Mutations may be selectable or unselectable . The rate of mutation can be enhanced by the
addition of chemicals called mutagens or by radiation. Auxotrophs are of particular use in
genetic analysis and as a basis for some bioprocess. Another useful class of mutants is condi-
tional mutants.
Gene transfer from one cell to another augments genetic information in ways that are not
possible through mutation only. Genetic recombination of different DNA molecules occurs
within most cells. Thus, genetic information transferred from another organism may become
a permanent part of the recipient cell. The three primary modes of gene transfer in bacteria
are transformation, transduction, and conjugation . Self-replicating, autonomous, extrachromo-
somal pieces of DNA called plasmids play important roles in transformation. Episomes , which
are closely related to plasmids, are the key elements in conjugation. Bacteriophages are
critical to generalized transduction, while temperate phages are the key to specialized transduc-
tion. Internal gene transfer can occur due to the presence of transposons , which probably also
play a role in the assembly of new plasmid.
We can use gene transfer in conjunction with restriction enzymes and ligases to genetically
engineer cells. In vitro procedures to recombine isolated donor DNA genes with vector DNA
(for example plasmids, temperate phages, or modified viruses) are called recombinant DNA
techniques . Once the vector with the DNA donor insert has been constructed, it can be moved
to a recipient cell through any natural or artificial method of gene transfer. Although trans-
formation is the most common technique in bacteria, a large variety of artificial methods have
been developed to insert foreign DNA into a host cell.
The application of recombinant DNA technology at the commercial level requires a judi-
cious choice of the proper host e vector system. E. coli greatly facilitates sophisticated genetic
manipulations, but process or product considerations may suggest alternative hosts. S. cere-
visiae is easy to culture and is already on the GRAS list, simplifying regulatory approval,
although productivities are low with some proteins and hyperglycosylation is a problem.
Pichia stipitis can produce very high concentrations of proteins, but the use of methanol pres-
ents challenges in reactor control and safety. Bacillus and the lower fungi may have well-
developed secretion systems that would be attractive if they can be harnessed.
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