Evolution and Genetic Engineering - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 705

Selection can be direct or indirect. An example of direct selection would be to find mutants
resistant to an antibiotic or toxic compound. A culture fluid containing 10 8 e 10 10 cells/mL is

subjected to a mutagenic agent. A few drops of culture fluid are spread evenly on a plate,
with the antibiotic incorporated into the gelled medium. Only antibiotic-resistant cells can
grow, so any colonies that form must arise from antibiotic-resistant mutants. If one in
a million cells has this particular mutation, we would expect to find about 10 e 100 colonies
per plate if 0.1 mL of culture fluid was tested.
Indirect selection is used for isolating mutants that are deficient in their capacity to
produce a necessary growth factor (e.g. an amino acid or a vitamin). Wild-type E. coli
grows on glucose and mineral salts. Auxotrophic mutants would not grow on such a simple
medium unless they were supplemented with the growth factor that the cell could no
longer make (e.g. a lysine auxotroph has lost the capacity to make lysine, so lysine must
be added to the glucose and salts to enable the cell to grow). The wild-type cell that needs
no supplements to a minimal medium is called a prototroph . Consider the selection of a rare
mutant cell that is auxotrophic for lysine from a population of wild-type cells. This cannot
be done directly, since both cell types would grow in the minimal medium supplemented
with lysine. A method that facilitated selection greatly is called replica plating (see Fig. 14.2 ).
A master plate using a lysine-supplemented medium will grow both the auxotroph and
wild-type cells. Once colonies are well formed on the master plate, an imprint is made
on sterile velveteen. The bristles on the velveteen capture some of the cells from each
colony. The orientation of the master plate is carefully noted. Then a test plate with
minimal medium is pressed against the velveteen; some cells at the point of each previous
colony then serve to inoculate the test plate at positions identical to those on the master
plate. After incubation (approximately 24 h for E. coli ), the test plate is compared to the
master plate. Colonies that appear at the same positions on both plates arise from wild-
type cells, while colonies that exist only on the master plate must arise from the auxotro-
phic mutants.
Another class of mutants is conditional mutants. Mutations that would normally be lethal
to the cell could not be detected by methods we have described so far. However, mutated
proteins are often more temperature-sensitive than normal proteins. Thus, temperature
sensitivity can often be used to select for conditionally lethal mutations. For example, the
mutant may be unable to grow at the normal growth temperature (e.g. 37 C for E. coli )
but will grow satisfactorily at a lower temperature (e.g. 25 C).
Mutation and selection have been used to tremendous advantage to probe the base
features of cell physiology and regulation. They also have been the mainstay of industrial
programs for the improvement of production strains. Mutation and selection programs
have been primarily responsible for increasing the yield of penicillin from 0.001 g/L in
1939 to current values of over 50 g/L in fermentation broth.

14.3. NATURAL MECHANISMS FOR GENE TRANSFER
AND REARRANGEMENT

We have learned that cells can evolve through mutation. In nature, cells can transform by
receiving genes from other organisms in their surroundings as well. Bacteria can gain and