Inserts amplification for knockout cassette construction

The goal of the study was to obtain optimal conditions for amplification of upstream and downstream genes fragments and introduce a cleavage site for restriction enzymes into them thanks to which it will be possible to clone fragments into a plasmid containing elements of the knockout cassette. The constructed cassettes will enable the removal of the tested genes from the C. albicans SC5314 genome using the SAT1-flipper method.
The deletion of tested genes encoding enzymes O-acetylhomoserine sulfhydrylases Met15p, homoserine O-acetyltransferases Met2p and cystathionine-γ-synthases Str2p enable the assessment of the impact of those enzymes on the phenotype of cells, on the virulence of the strain, and allows to recognize the in vivo function of the tested proteins in L-methionine biosynthesis.
The studies were financially supported from the project OPUS 20 financed by the National Science Centre (No. UMO-2020/39/B/NZ7/01519).

本研究旨在获得扩增上下游基因片段的最优条件，并在片段中引入限制性内切酶切割位点，从而可将片段克隆至含敲除盒元件的质粒中。构建的敲除盒将通过SAT1-flipper法实现从白色念珠菌（C. albicans）SC5314基因组中去除目标基因。删除编码O-乙酰高丝氨酸巯基酶Met15p、高丝氨酸O-乙酰转移酶Met2p及胱硫醚-γ-合酶Str2p的目标基因，能够评估这些酶对细胞表型及菌株毒力的影响，并有助于揭示目标蛋白在L-甲硫氨酸生物合成中的体内功能。本研究得到国家科学中心（National Science Centre）资助的OPUS 20项目（编号：UMO-2020/39/B/NZ7/01519）支持。