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Selections

©2000 written by Gary Roberts, edited by Timothy Paustian, University of Wisconins-Madison

II C. SELECTIONS

In selections, one sets up conditions so that only the cells displaying the desired phenotype prosper. In practice, this means that only cells with the desired phenotype give rise to colonies on the "selective" agar plate. It is the range of genotypes which cause that phenotype that affects the actual frequency of different classes of survivors in the selection. You should try to put yourself in a position to do a selection (as opposed to a screen or enrichment) because your hunt will be faster (you must analyze only those strains of the desired phenotype) and because it uses the power of genetics, in that you can demand very rare events. If you have a good selection, you need not mutagenize. This tactic avoids double mutants (strains with two or more mutations) but, by relying on spontaneous mutants, you may well isolate the same genotype again and again. Recognize that selection are for living organisms with a selectable phenotype. Depending on the situation, this phenotype might have arisen through mutation or recombination.

As an aside, it is often forgotten that every growth condition is selective: each selects against mutants that grow less well than the rest and for mutants that grow better than the rest under those conditions. For example, a mutation that confers both phage resistance and poor growth will be selected for when phage are present and selected against when phage are absent from the growth conditions.

Some selections are "stronger" or "tighter" than others: reversion of an auxotroph on minimal medium is a strong selection, since only prototrophs will grow; growth in nutrient medium is a weak selection, since the "best" strains will only grow slightly better than the rest. On the other hand, such a selection can be a serious concern in those cases of strain storage where cells are inoculated onto the surface (a "slant") or into the interior (a "stab") of a nutrient agar plug in a vial and then stored at temperatures where some growth can occur. A preferable method of strain maintenance is -80°C in a cryoprotective agent like 10% DMSO or glycerol.

A selection always provides strains of the demanded phenotype, but not necessarily of the desired genotype. You will therefore typically need to further analyze the generated strains to verify their genetic makeup.

II D. OTHER COMMENTS ON SCREENS AND SELECTIONS

As described in section III F5, there are cases where it may take a period of time before the mutant phenotype is expressed. Such factors need to be taken into account in the actual timing and conditions employed in a particular screen.

Some hunts work differently at different stages of cell growth. A chemical that might have a profound effect on spore germination might have a very different effect if vegetative cells are used in the analysis. In general, the permeability and growth requirements of microorganisms change rather dramatically depending on the stage of the growth cycle. Such considerations are particularly important for those prokaryotes that go through a more dramatic form of differentiation like sporulation.

As will be discussed further in section III F5, one can guess the frequency of an event and the likelihood of its detection in a given selection or enrichment based on one's understanding of the affected genes and their products. If in reality one sees a frequency of occurrence that is very different, this is a strong suggestion that either one's perception of the underlying genetic and biochemical reality is incorrect or something curious happened in the selection itself. In general, selections and screens nearly always work, in that they give you isolates of the demanded, and expected, phenotypes. On the other hand, these mutants may not be arising because of the desired genotype. Arguably, it is the essence of genetics to discern the genotypic nature causing a given phenotype.

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