Journal: PLoS Pathogens
Article Title: Gene Annotation and Drug Target Discovery in Candida albicans with a Tagged Transposon Mutant Collection
Figure Lengend Snippet: Construction of tagged transposon mutants in C. albicans . (A) A commercial Tn5 transposon was modified by inclusion of a Gateway conversion cassette (with the ccdB selection gene and chloramphenicol resistance gene (Cm R )), a kanamycin resistance gene (Kan R ), and the UAU1 marker cassette at the multiple cloning site (MCS). A reaction with the modified Tn5, a pool of TagModules, and LR clonase induces recombination at the att sites, placing the TagModule within the transposon mosaic ends (Tn5L and Tn5R). (B) To generate the heterozygous disruption strains, multiple genomic DNA libraries were constructed and then mutagenized in vitro with the pool of tagged Tn5 transposons from (A). Plasmids containing individual insertions were recovered from E. coli and sequenced (arrow) to determine the disrupted gene and its corresponding tag. Results were sorted to maximize the unique gene insertions/tag pairs. Select insertions were amplified, followed by excision of the genomic DNA containing the tagged transposon insertion. Transformation of these inserts into C. albicans was mediated by homology of the genomic sequence flanking the transposon insertion, and integration was selected for based on the arginine prototrophy conferred by the Arg+ marker. Homologous recombination results in a gene disruption with a tagged Tn5 transposon. (C) Screening methodology. Approximately equivalent numbers of tagged transposon mutants are combined to create a tagged pool. Pools are then screened in diverse conditions, generally over 20 generations of competitive growth with pools being harvested and diluted every 5 generations (5 g, 10 g, 15 g, 20 g). The genomic DNA is then extracted from the pool and the uptags and downtags amplified separately using the common priming sites. After the pooled growth assay, the amplicons are then hybridized to an Affymetrix TAG4 microarray to calculate tag intensity as a proxy for strain abundance.
Article Snippet: Correct clones were then transferred to the pAG416GPD-ccdB destination vector using the LR clonase reaction (Invitrogen), and resequenced as described.
Techniques: Modification, Selection, Marker, Clone Assay, Construct, In Vitro, Amplification, Transformation Assay, Sequencing, Homologous Recombination, Growth Assay, Microarray