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

9703527达席尔瓦利用酿酒酵母生产蛋白质受到可用于基因传递的载体选择狭窄的限制。目前，可以用低拷贝数的质粒导入单基因。当通过增量介导的传递传递多个拷贝时，多个拷贝被串联插入，从而导致不稳定。目前还没有可用于控制多个基因或多个非串联拷贝添加到酿酒酵母的方法。这项SGER提案将测试使用酵母转座作为外源基因载体的可行性。酵母转座是一种可移动的DNA片段，通过类似逆转录病毒的机制进行复制。将构建一个盒，该盒包含感兴趣的基因(在这种情况下，为了便于分析，包含β-半乳糖苷酶基因)和一个选择基因，该选择基因可用于选择转座的酵母细胞，后者通过“弹出”机制被删除，因此盒和相同的标记可用于插入另一个基因。URA3基因两侧有两个直接的细菌His G重复序列，将被用作开关。转座的细胞将被选在尿嘧啶缺乏的平板上，然后被“弹出”的细胞将被选在5-FOA上，这只允许那些没有URA3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. ***