|[Previous] | [Next]|
Table of Contents
Why Do Genetics
More Basic Concepts
Mutagenesis in vitro
Effects of Mutations
Two Factor Crosses
Other Mapping Methods
Problem Set 1
Problem Set 2
Search | Send us your comments
©2000 written by Gary Roberts, edited by Timothy Paustian, University of Wisconins-Madison
II. BASIC GENETIC METHODS
Successful genetic analysis demands that you be able to recognize and isolate the desired mutants at a frequency comparable to that of their generation (described in Section III). The range of techniques that are of use for this are screens, enrichments, and selections. These tools are relevant to both classical and inverse genetic approaches since you can only identify mutants that occur at or above your level of detection, regardless of their mode of generation. Even in inverse genetics, many of the products of a reaction/mutagenesis will not be the desired class.
II A. SCREENS
A screen is the analysis of different bacterial isolates for a given phenotype or property (like unusual growth, the level of a given enzyme, the presence of an interesting metabolite, the level of a particular antigen, or the presence of a region of DNA capable of hybridizing to a given probe). It should not be confused with a selection ,which is a demand for a given phenotype and is therefore orders of magnitude more "powerful". As an example, you would select for Rifr resistance by plating cells on rifampicin (only Rifr cells will give rise to colonies); you would screen for Rifs by replica-printing as described below, because there is no growth conditions where only Rifs cells will grow. As this example shows, a screen is typically used when the "growth properties" of the desired mutants is either unaffected or deleteriously affected in comparison with that of wild type. Section III will discuss the frequency with which various sorts of mutations occur, but it is equally important to consider one's ability to detect various events. Obviously it is necessary to have a means of detecting desired mutant classes with at least the frequency of their occurrence in the population. The numbers themselves are crucial (if a little vague) and clearly the most painful or difficult screens are those that involve a separate analysis, typically biochemical, of each member of the population to be analyzed. Easier screens are those where a large number can be analyzed in a single assay.
Tough screens. If one were searching for a mutant defective in a given enzymatic activity, this could be done by picking a number of single colonies, growing each up in liquid, breaking the cells in each case and assaying for the enzyme in question. Screens for bacterial isolates that produce altered levels or types of secondary metabolites are often so performed and are typically rather difficult for that reason. These screens are so difficult that one can really only consider doing between 101 and 104 separate assays and therefore one must either mutagenize heavily with chemical mutagens or use selectable transposons in order to generate mutants at a frequency that allows detection.
Easier screens. One of the easiest screens involves replica printing. In this system one grows up between 10 0 and 1000 single bacterial colonies on a plate and prints this onto velveteen. Small numbers of cells from each colony adhere to the velveteen which is then used to imprint this pattern onto various other media. What one gets is an image of the original "master" plate superimposed on these other media so that a bacterial colony unable to grow on a certain media can be found. With this screen one can analyze approximately 500 colonies per plate and a 200 plate experiment is not overly formidable so that up to 105 colonies can be screened easily.
Another form of screen involves suboptimally supplemented plates. In this case the media is appropriately supplemented so that mutants that require a particular supplement (which is present at only very low level) grow in very flat, distinctive colonies, because they spread themselves over the agar surface to get the trace supplement. In this way approximately 1000 colonies can be screened on a plate and those of appropriate morphology chosen and retested. Again, approximately 104 to 105 colonies can easily be screened. Color indicator plates can also be extremely useful. Examples of these are plates where a particular chemical is incorporated into the cell and, if a given enzyme is present, that chemical is processed to a colored compound. Mutants defective in the production of that enzyme function will remain uncolored and can be detected. Similarly, pH indicator plates have been used for scoring sugar utilization. The efficacy of such plates is a function both of the diffusion of the color as well as the experimenter's ability to detect it. Typically 103 to 104 colonies can be screened per plate giving an ability to detect mutants arising at 10-5 to 10-6 (when 100 plates are analyzed).
Finally, there are very powerful molecular biological methods that involve either radiolabeled nucleic acid probes or antibodies to a given protein ("tagged" in some way) being used to screen either bacteriophage plaques or colonies. In general, this method relies on the plaque or the colony being "printed" to a membrane that is then examined for its ability to hybridize to a particular nucleic acid probe or be recognized by a antibody. Some screens of this sort can be extremely powerful, allowing the screening of more than 106 plaques or colonies on a single plate.
General considerations. A major consideration is the destructiveness of your assay. If your assay involves killing the cells of interest, then you must in some way replicate your "master" because your goal will always be to isolate an organism with a given trait. To be of any use, that organism must be alive. Many of the screens described above, especially the latter ones, are destructive. A second point is the use of pools of organisms (as opposed to examining isolates one at a time). If a mutant of a given phenotype is detectable even when present in only a low percentage of the population, one can greatly enhance one's screening in a two-step procedure: pool potential mutants and analyze the various pools for the phenotype sought. One would then return to any "successful" pool and examine individual isolates for those that caused the pool to give a positive response. This system obviously allows the screening of many more potential mutants than a system based on the analysis of homogeneous colonies or liquid cultures.
II B. ENRICHMENTS (counter-selections).
Typically enrichment procedures rely on finding a condition where the rare desired organisms do not grow, but the majority class does. One then adds something to the media to kill the growing bacteria and finally screens the non-growing, but living, survivors. Some typical methods involve killing the growing cells with DNA analogs or chemicals which affect cell wall synthesis, for example, penicillin or cycloserine. With both of these systems, a serious concern is whether or not your desired bugs will grow enough to die either because they are being fed by the growing cells or by the recently killed cells. The point is that one is running a gauntlet between killing the growing cells efficiently and not killing the desired organisms. This gauntlet means that such enrichments typically yield no more than 101 to 103 fold success. They can be repeated, but this gives rise to more siblings (see section III G3).
[Previous] | [Next]