Improved efficiency and stability of multiple cloned gene insertions at the delta sequences of Saccharomyces cerevisiae
Improved efficiency and stability of multiple cloned gene insertions at the delta sequences of Saccharomyces cerevisiae
复制标题
DOI：
10.1007/s002530051059
复制
发表时间：
1997-09-01
影响因子：
5
通讯作者：
DaSilva, NA
中科院分区：
文献类型：
--
作者：
Lee, FWF;DaSilva, NA
通过同源重组将诱导型表达盒（酵母CUP 1启动子融合到大肠杆菌lacZ结构基因，CUP 1 p-lacZ）和细菌新霉素抗性基因（neo）插入酿酒酵母基因组中，对两种δ整合载体进行了评价。通过对氨基糖苷类G418的抗性选择含有整合的细胞。第一个载体是仅含有一个δ序列的传统构建体;使用该载体，转化效率和每个细胞的整合数相当低。第二个携带两个delta序列侧翼所需的插入，和不需要的细菌序列被删除的限制酶消化转化前立即。当使用这种双δ载体时，整合的拷贝数相对于单δ系统增加了一倍以上，并且最终的β-半乳糖苷酶水平超过了用基于2 μ的质粒获得的水平。此外，整合出现更稳定的长期连续培养（有和没有诱导的lacZ基因）比那些通过单一的δ载体。
Two delta-integration vectors were evaluated for the insertion of an inducible expression cassette (the yeast CUP1 promoter fused to the Escherichia coli lacZ structural gene, CUP1p-lacZ) and a bacterial neomycin-resistance gene (neo) into the genome of Saccharomyces cerevisiae via homologous recombination. Cells containing integrations were selected by resistance to the aminoglycoside G418. The first vector was a traditional construct containing only one delta sequence; with this vector, the transformation efficiency and the number of integrations per cell were quite low. The second carried two delta sequences flanking the desired insert, and the unneeded bacterial sequences were removed by restriction-enzyme digestion immediately before transformation. When this double delta vector was employed, the integrated copy number was more than doubled relative to the single delta system and final beta-galactosidase levels exceeded those obtained with the 2 mu-based plasmid. Furthermore, the integrations appeared more stable in long-term sequential culture (both with and without induction of the lacZ gene) than those obtained via the single delta vector.