pDelcas plasmid vector質(zhì)粒載體

Gene deletion in Schizophyllum commune is hampered by a low incidence of homologous integration. As a consequence, extensive screening is required to identify a transformant with the desired genotype. To alleviate this and to facilitate the assembly of deletion plasmids, vector pDelcas was constructed. This construct has a set of restriction sites, which allows for directional cloning of the flanking sequences at both sides of a nourseothricin resistance cassette. Moreover, it contains a phleomycin resistance cassette elsewhere in the plasmid, which is used to screen for transformants with an ectopic integration of the pDelcas derivative. The use of pDelcas derivatives in combination with an improved PCR screening protocol permitted the efficient identification of S. commune deletion strains. This procedure may also function in other basidiomycetes.

Vector pDelcas is based on a pUC20 backbone. It contains a phleomycin and a nourseothricin resistance cassette. Van91I and SfiI sites flank fragments from bacteriophage λ of 2,4 and 0.64 kb, respectively (indicated in bold). Custom made sticky ends in these sites allow directional cloning when combining the primers depicted in the lower panel. The underlined sequence of the primers represent the recognition site of SfiI, while the N18 region represent the flank specific sequence

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