Amen Triana, Kaganovich Daniel
Department of Cell and Developmental Biology, Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel.
Microb Cell. 2017 Jun 5;4(6):182-190. doi: 10.15698/mic2017.06.576.
We present a set of vectors containing integrative modules for efficient genome integration into the commonly used selection marker loci of the yeast . A fragment for genome integration is generated via PCR with a unique set of short primers and integrated into , , , and loci. The desired level of expression can be achieved by using constitutive (, ), inducible (, ), and daughter-specific () promoters available in the modules. The reduced size of the integrative module compared to conventional integrative plasmids allows efficient integration of multiple fragments. We demonstrate the efficiency of this tool by simultaneously tagging markers of the nucleus, vacuole, actin, and peroxisomes with genomically integrated fluorophores. Improved integration of our new pDK plasmid series allows stable introduction of several genes and can be used for multi-color imaging. New bidirectional promoters (, , and ) allow tractable metabolic engineering.
我们展示了一组载体，其包含用于高效基因组整合到酵母常用选择标记基因座的整合模块。通过使用一组独特的短引物进行PCR生成用于基因组整合的片段，并将其整合到、、和基因座中。通过使用模块中可用的组成型（、）、诱导型（、）和子代特异性（）启动子，可以实现所需的表达水平。与传统整合质粒相比，整合模块尺寸减小，从而允许多个片段的高效整合。我们通过用基因组整合的荧光团同时标记细胞核、液泡、肌动蛋白和过氧化物酶体的标记物，证明了该工具的效率。我们新的pDK质粒系列改进的整合允许稳定引入多个基因，并且可用于多色成像。新的双向启动子（、和）允许进行易于处理的代谢工程。
