1887

Abstract

We have developed a system for the induction of marker-free mutation of . The system features both the advantages of the use of antibiotic-resistance markers for mutant selection, and the ability to efficiently remove the markers, leaving unmarked mutations in the genome. It utilizes both a selective marker cassette and a counter-selective marker cassette. The selective marker cassette contains a chloramphenicol-resistance gene and the gene, which encodes the repressor for the arabinose operon () of . The counter-selective marker cassette consists of a promoterless neomycin (Nm)-resistance gene () fused to the promoter. First, the chromosomal locus is replaced with the counter-selective marker cassette by double-crossover homologous recombination and positive selection for Nm resistance. The selective marker cassette is connected with upstream and downstream sequences from the target locus, and is integrated into the upstream region of the target locus by a double-crossover event. This integration is also positively selected for, using chloramphenicol resistance. In the resultant strain, AraR, encoded by on the selective marker cassette, represses the expression of in the absence of -arabinose. Finally, the eviction of the selective marker cassette together with the target locus is achieved by an intra-genomic single-crossover event between the two downstream regions of the target locus, and can be selected for based on Nm resistance, because of the excision of . The counter-selective marker cassette remaining in the genome, whose expression is switched on or off based on the excision or introduction of the selective marker cassette, is used again for the next round of deletion. Using this system, the 3.8 kb region and the 41.8 kb region have been efficiently and successfully deleted, without leaving markers in the target loci. The positive selection and simple procedure will make it a useful tool for the construction of multiple mutations.

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2008-09-01
2020-07-09
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