Methods:Strain construction strategies
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This page reviews some of the kinds of strain construction strategies used by E. coli molecular geneticists, and discusses how strain construction can be used to address specific kinds of biological questions.
Loss of function mutations are important for identifying the essential components for any biological process. Several kinds of loss-of-function or "knockout" mutations are available for E. coli. These can broadly be divided into classes:
Systematic deletions from the Keio collection
Hirotada Mori and coworkers used recombineering methods to generate knockout mutations in annotated genes. These mutations replace the gene with an antibiotic resistance cassette (kanamycin resistance for the original Keio collection) in a way that should not be polar on downstream genes. A second generation of systematic mutations inlcudes alternative drug resistances and tags each insertion with an oligonucleotide signature that can be followed by sequencing or hybridization.
Keio deletions-insertions are designed to allow removal of the antibiotic resistance cassette by site-specific recombination between flanking frt sites. The Keio collection includes some mutations in essential genes, presumably due to selection for local duplications.
Because they are marked with antibiotic resistance genes, Keio deletion-insertions can be moved into new strain backgrounds by selection for the resistance marker. The markerless deletions made after FRT recombination can be moved by conjugation or transduction using linked markers.
A large number of deletions that remove multiple genes have been described in the literature. Large deletions can be moved into new strains by transduction or conjugation. This can be done using either a marker linked to one or both deletion endpoints, or by selection for loss of a marker covered by the deletion in the recipient cell.
See also: PEC database
Libraries of transposon mutations remain a convenient way to look for genes involved in a process. Screens or selections based on transposon insertions are often easier than constructing the desired genetic background with all of the Keio knockouts. Several transposon mutagenesis systems are available. Desirable features in transposon mutagenesis systems include provision of transposase in trans to generate stable insertions that are not able to transpose to another location during later manipulations, and engineered transposons that make the insertion locations more random.
Loss of function can also be examined using nonsense mutations. Nonsense mutants have the advantage of being conditional in the presence or absence of appropriate suppressor tRNAs.
Stable knockouts are not just problematic for essential genes. Stable knockouts give terminal phenotypes, but conditional mutations often provide additional information about when and where a gene product functions in a process. Ideally, one would like to have a way to instantaneously remove a gene product of interest; this is not technically possible, so a variety of techniques are used to approximate this situation.
- ts and cs mutations.
- conditional loss of the gene
- chemical genetics - addition of inhibitors
- conditional expression of inhibitors of expression or activity of the product
Moving mutant alleles
In cases where a mutant allele is not directly selectable, there are two basic ways to transfer the mutation into a desired genetic background.
- replace a marked version of the gene of interest with the desired allele
- cotransduce the desired allele with a linked marker
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