Reece-Hoyes John S, Walhout Albertha J M
Cold Spring Harb Protoc. 2018 Jul 2;2018(7):2018/7/pdb.prot094961. doi: 10.1101/pdb.prot094961.
Generating DNA-bait strains for gateway-compatible yeast one-hybrid (Y1H) screens involves three steps. The first is to generate an Entry clone containing the DNA-bait of interest. Gateway cloning is used to clone larger baits, such as promoters, into pDONR-P4-P1R. (An alternative set of steps is also presented in this protocol that describes the creation of Entry clones by annealing primers and performing conventional ligation into pMW#5-a strategy best suited for smaller DNA-baits up to 100 bp.) The second is to transfer this DNA-bait from the Entry clone to the two Y1H reporter Destination vectors, pMW#2 () and pMW#3 (). A two-step process is used because Entry clones generate a versatile resource that can be used for transfer of DNA-baits into a variety of vectors, for instance, upstream of the green fluorescent protein-encoding ORF to study spatiotemporal expression patterns. The final step is to integrate the and reporter constructs into the genome of the Y1H yeast strain, YM4271. The entire process takes 24-32 d, plus sequence confirmation if necessary.
为适用于Gateway技术的酵母单杂交（Y1H）筛选生成DNA诱饵菌株涉及三个步骤。第一步是生成一个包含感兴趣的DNA诱饵的入门克隆。Gateway克隆用于将较大的诱饵（如启动子）克隆到pDONR-P4-P1R中。（本方案还介绍了另一组步骤，即通过引物退火并进行常规连接到pMW#5中来创建入门克隆——这是最适合长度达100 bp的较小DNA诱饵的策略。）第二步是将该DNA诱饵从入门克隆转移到两个Y1H报告载体pMW#2（ ）和pMW#3（ ）中。采用两步法是因为入门克隆可生成一种通用资源，可用于将DNA诱饵转移到多种载体中，例如，转移到绿色荧光蛋白编码开放阅读框的上游以研究时空表达模式。最后一步是将 和 报告构建体整合到Y1H酵母菌株YM4271的基因组中。整个过程需要24 - 32天，如有必要还需进行序列确认。
