A general approach to the analysis of a bacterial system

©2003 written by Gary Roberts, edited by Timothy Paustian, University of Wisconins-Madison

IX D. A GENERAL APPROACH TO THE GENETIC ANALYSIS OF A BACTERIAL SYSTEM

Thus far, the text has considered both good and bad approaches to solving genetic problems. The section following is a suggestion as to what elements one might want to use in the best of all possible worlds for the genetic analysis of a new bacterial system. The tools needed might be:

1. A system of mutagenesis in vivo, presumably employing both chemical and transposon mutagenesis.

2. A good screening system for the phenotype(s) of interest. For example, the identification of appropriately suboptimally supplemented plates would be useful. The requirement for "plates" or solid media is important since it allows examination of hundreds of isolated, potentially different mutants on each plate. You cannot examine enough different isolates in separate liquid cultures.

3. An enrichment scheme for the phenotypes of interest.

4. A system for genetic transfer in and out of the strain in vivo: this would typically entail conjugative plasmids capable of moving into the strain as well as plasmids capable of replicating within the strain.

5. Methodology for transformation allowing the use of all manner of replicons constructed, manipulated or mutagenized in vitro.

6. Niceties for studying regulation include the ability to assay reporter genes fused to a gene of interest. This can be powerful in assaying regulation and in demanding altered regulatory mutants (see Section XI).

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