Chapter 12 : Gene Replacement Systems

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With the completion of the genome sequence, the focus of research has turned to functional characterization of genes, and, in particular, identification of virulence factors. The inability to identify double-crossover events was a major impediment in the study of pathogenicity. The first mutant of the complex made through allelic replacement was a BCG mutant, created using the gene as a target. A major factor in the small number of mutants obtained using suicide plasmids was the efficiency of transformation. This difficulty is avoided by using replicating plasmids, although there is the new problem of ensuring plasmid loss. The first counterselectable marker to be described in mycobacteria was streptomycin sensitivity. This system takes advantage of the fact that the S12 ribosomal protein is the target of streptomycin. A major drawback when using replicating plasmids was that it was necessary to isolate plasmid free cells. To induce efficient plasmid loss at will, a thermosensitive replicon was isolated. Marked mutants, in which an antibiotic resistance gene is used to interrupt the gene of interest, are the most straightforward to make. Even when the gene studied is essential, studying the phenotype of a conditional mutant can be useful because it can help to unravel the function of the gene. In conclusion, many genetic tools are now available for studying mycobacteria. It is possible to select directly for allelic exchange in a single-step strategy or to construct an unmarked mutation in a two-step strategy.

Citation: Stoker N, Sander P, Reyrat J. 2005. Gene Replacement Systems, p 183-190. In Cole S, Eisenach K, McMurray D, Jacobs, Jr. W (ed), Tuberculosis and the Tubercle Bacillus. ASM Press, Washington, DC. doi: 10.1128/9781555817657.ch12
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Figure 1

Two-step strategy for allelic replacement. Positive selection of allelic exchange mutants in a two-step selection strategy, using a counterselectable marker, is shown. CSM, counterselectable marker; SM, selectable marker; wt, wild-type allele; mut, mutated allele. Adapted from reference 54 with permission.

Citation: Stoker N, Sander P, Reyrat J. 2005. Gene Replacement Systems, p 183-190. In Cole S, Eisenach K, McMurray D, Jacobs, Jr. W (ed), Tuberculosis and the Tubercle Bacillus. ASM Press, Washington, DC. doi: 10.1128/9781555817657.ch12
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