摘要
转录因子及启动子是基因回路的基础。相较于原核启动子,真核启动子作用机制复杂,增加了全新设计与改造的难度。目前有限数量的真核转录因子及启动子成为在哺乳动物细胞中设计并实现复杂基因回路以满足各类临床或工业应用需求的瓶颈。文中介绍了基于能够结合特定DNA序列的DNA结合结构域,通过柔性连接肽连接到转录抑制模块KRAB,构建抑制型转录因子以及通过在SV40启动子下游插入结合序列构建对应启动子的方法。而后,在哺乳动物细胞系中通过流式细胞术对其抑制转录的强度、不同转录因子及启动子对之间的正交性进行了测定。文中提供了一套标准化的、可调节的转录因子及启动子的全新设计与构建方案。基于该方案所构建的5对抑制型转录因子及启动子对能够在哺乳动物细胞中起到不同程度的抑制效果且相互正交。文中构建的哺乳动物转录因子及启动子对扩充了哺乳动物生物元件库,为构建复杂真核基因回路打下了基础;运用该设计方法能够根据需求构建更多正交的人工转录因子及启动子对。
Transcriptional regulation is crucial for regulated gene expression.Due to the complexity,it has been difficult to engineer eukaryotic transcription factor (TF)and promoter pairs.The few availabilities of eukaryotic TF and promotor pairs limit their practical use for clinical or industrial applications.Here,we report a de novo construction of synthetic inhibitory transcription factor and promoter pairs for mammalian transcriptional regulation.The design of synthetic TF was based on the fusion of DNA binding domain and Kruppel associated box transcription regulating domain (KRAB).The synthetic promoter was constructed by inserting the corresponding TF response element after SV40promoter.We constructed and tested five synthetic inhibitory transcription factor and promoter pairs in cultured mammalian cells.The inhibition capability and orthogonality were verified by flow cytometry.In summary,we demonstrate the feasibility of constructing mammalian inhibitory TF and promoter pairs,which could be standardized for advanced gene-circuit design and various applications in the mammalian synthetic biology.
作者
杨子杰
潘祎杰
蔡毅鸣
傅彤
冯骜
刘燕
王一恒
熊心旋
蔡亮
Zijie Yang;Yijie Pan;Yiming Cai;Tong Fu;Ao Feng;Yan Liu;Yiheng Wang;Xinxuan Xiong;and Liang Cai(School of Life Sciences,Fudan University,Shanghai 200438,China)
出处
《生物工程学报》
CAS
CSCD
北大核心
2018年第12期1886-1894,共9页
Chinese Journal of Biotechnology
基金
教育部"基础学科拔尖学生培养计划"研究课题经费(Nos.20180202
20160804)
复旦大学本科生学术研究资助计划(FDUROP)资助~~
关键词
真核转录调控
人工转录因子
人工启动子
合成生物学
生物元件
eukaryotic transcription regulation,synthetic transcription factor,synthetic promoter,synthetic biology,Bio-Brick