Mutant collection

Principle and Method :

Each CDS (1), defined during the annotation process has been replaced by a cassette containing the gene for kanamycin resistance under the control of a strong promoter (Phage T5 promoter) by homologous recombination. The extremities of the integration cassette, called "recombination footprints" consist of the specific regions which frame the gene to be mutated. The replacement usually covers the totality of the ORF (2). However, to avoid altering essential regions such as the RBS (3)) or the promoters of neighboring genes, we have had to construct a replacement in which the target ORF is truncated by several amino acids. The molecular construction of the cassettes is obtained in detail using PCR(4) which recombines the recombination footprints with the gene for antibiotic resistance. After transformation of Acinetobacter baylyi with each of these cassettes, the mutants are selected on a mineral medium supplemented with succinate and kanamycin.

Technique :

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Production phase:

Optimization of the transformation protocol and organization of the "pipeline" has permitted us to attain a success rate of over 90%, and a capacity of 400 mutants initiated every two weeks. The complete cycle for the production of a mutant from the transformation step to storage is 4 weeks. This cycle includes control PCRs to check on the replacement of the ORF by the integration cassette and systematic sequencing of the genomic regions of the mutant which are adjacent to the integration cassette.

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Results :

Of the 3205 genes annotated in A. baylyi which are candidates for mutation, we have attempted to inactivate 3061 genes We produced replacement mutants for 2621 genes, which represents 77% of the genes of A. baylyi. For 96% of the knock-outs, two independent clones were obtained.

Distribution of mutants according to functional category of the genes

Perspectives :

The systematic gene replacement phase which has now been achieved will be extended by a targeted finishing phase, to attempt to obtain mutants in genes in which the first attempt has failed. Simple repeat of the experiments should suffice for these "technical failures;" new primers in more favorable regions will be selected for some of them. We will also try to repeat the experiments on more enriched media in order to obtain mutants of the genes which are essential on succinate.

An analytic phase will now be undertaken in the collection, in addition to systematic phenotyping in order to study, for example, genes with unknown function which co-localize with genes which participate in the same metabolic pathway.

Glossary :

(1) CDS : Coding DNA Sequence.
(2) ORF : Open Reading Frame.
(3) RBS : Ribosome Binding Sequence.
(4) PCR : Polymerase Chain Reaction.

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