영문 실험 레포트입니다..(조금은 허접합니다.. 참고만 하세요..)
gene disruption using URA-blaster..
MATERIAL AND METHODS
The outer cell wall of Candida albicans consists of highly branched glycoproteins modified by N-linked glycosylation including mannosylphosphorylation. Mannosyl-phosphate confers a net negative charge to the cell wall of C. albicans, resulting in binding a positively charged dye Alcian Blue. Previous studies have proposed that mannosylphosphorylation involves in virulence of C. albicans via modification of the outer cell surface. Saccharomyces cerevisiae MNN4 which is required for the mannosylphosphate transfer has high homology with that of C. albicans. The MNN4 gene of C. albicans was disrupted by using Ura-blaster system which was constructed based on the pDH2 plasmid. To select ura- strains, a plate containing 5-fluoroorotic acid was used. After the secondary round transformation, null mutant strains were screened by PCR analysis. The effect of heterozygous mnn4∆ mutants on the cell wall was analysed by Alcian Blue binding assay. The results addressed that homologous mnn4∆ mutant strains showed a significant loss of Alcian Blue binding.
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