SGER: Advanced Ty-Mediated Integration Methods for Metabolic Engineering and Protein Production in Yeast

9703527 DaSilva使用Saccharomyces gallaae生产蛋白质受到可用于基因递送的载体选择狭窄的限制。 目前，质粒可用于以低拷贝数引入单基因。 当通过δ介导的递送递送多个拷贝时，多个组串联插入，导致不稳定性。 目前还没有一种方法可以用于控制添加多个基因或多个非串联拷贝到S。啤酒。 这个SGER计划将测试使用酵母转座（通过类似逆转录病毒的机制复制的移动的DNA片段）作为外源基因载体的可行性。 将构建含有目的基因（在这种情况下为β-半乳糖苷酶基因以便于测定）加上选择基因的盒，所述选择基因可用于选择转座的酵母细胞，并且后者通过“弹出”机制缺失，因此所述盒和相同的标记可用于插入另一个基因. 侧翼为两个直接细菌his G重复序列的URA 3基因将用作开关。 转座的细胞将在尿嘧啶缺陷平板上选择，然后已经“弹出”的细胞将在5-FOA上选择，5-FOA仅允许那些没有URA 3基因的细胞生长。 ***

9703527 DaSilva The use of Saccharomyces cerevisiaae to produce proteins is limited by the narrow choice of vectors available for gene delivery. Currently, plasmids can be employed to introduce single genes at low copy number. When multiple copies are delivered by delta mediated delivery, the multiple sets are inserted in tandem leading to instability. There is no method available currently that can be used for the controlled addition of multiple genes or multiple, non- tandem copies to S. cerevisiae. This SGER proposal will test the feasibility of using yeast transposes, mobile segments of DNA that replicate by a retrovirus-like mechanism, to act as vectors for foreign genes. A cassette will be constructed that contains the gene of interest (in this case the gene for beta- galactosidase for ease of assay) plus a selection gene that can be used to select transposed yeast cells and latter deleted by a "popping out" mechanism so the cassette and same marker can be used to insert another gene. The URA3 gene flanked by two direct bacterial his G repeats will be used as the on-off switch. The transposed cells will be selected on uracil-deficient plates and then the cells that have been "popped-out" will be selected on 5-FOA which allows only those cells without the URA3 gene to grow. ***