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Basic summary on knock-outs In vitro a DNA fragment is altered by insertion of a fragment containing a selectable marker The interrupted gene is introduced into the cells In some cells homologous recombination results in the replacement of the resident gene with the interrupted gene Homozygous transgenic cells are often referred to as knock-outs These knock-outs can then be examined to determine what if any phenotype is associated with mutations in the targeted gene If a knock-out phenotype is found then the locus responsible for the mutation can be placed on a genetic map by standard crossing and segregation analysis The knock-out strategy can be used to test whether an isolated gene functions as thought Also panels of knock-out mutants can be created by random insertion of transposable elements An important mechanism involved in the regulation of bacterial gene expression in response to environmental signals is the use of alternative sigma factors to alter RNA polymerase specificity Ecoli and other bacteria often encounter nutrient limitations resulting in periods of negligible or absent growth E coli and other bacteria respond to starvation by entering a metabolic state known a stationary phase This allows them to survive environmental stresses such as oxidative stress heat high salt and UV radiation In order to generate a mutation in the rpoS gene allelic exchange may be carried out by the use of a suicide vector Disruption of chromosomal rpoS DNA by a fragment conferring some sort of antibiotic resistance can be achieved by homologous recombination by a plasmid To construct this plasmid two separate cloning steps can be performed The insertion mutation can be obtained by ligating a DNA fragment from a vector encoding for antibiotic resistance into the rpoS The ligation product can then be introduced in to the suicide plasmid mentioned in the beginning at certain restriction sites that would be present within the multiple cloning site of the plasmid The resulting plasmid confers
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