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人工转录装置可以按照所设计的方式在原核细胞和真核细胞中调节基因表达。本研究在大肠杆菌中设计并构建了一种由合成启动子和人工转录因子构成的人工转录装置:其中,合成启动子包含了一种弱启动子Plac突变体与位于其上游的λ噬菌体操纵区(O_R2或O_R3)的不同组合,而人工转录因子则利用细菌RNA聚合酶α亚基的N端和λ噬菌体CI蛋白的C端组成的融合蛋白α-CI作为效应域,CI蛋白作为DNA结合结构域。利用绿荧光蛋白作为报告基因对这一人工转录装置检测发现,它可以成功开启报告基因的转录活性。随后通过改变这一人工转录装置的某些参数,例如,增加启动子上游操纵序列的拷贝数、使用温敏型CI取代野生型CI、在系统中引入CI的阻遏蛋白Cro等多种方式,实现了对报告基因转录强度的精细调控。这一人工可调控转录装置可以在原核细胞中实现对报告基因表达强度的精细调控,在原核细胞中调节基因线路的信号输出强度方面具有一定的应用前景。
Abstract：Artificial transcription devices can regulate gene expression as designed in both prokaryotes and eukaryotes. In this study, an artificial transcription device made up of a synthetic promoter and an artificial transcription factor was constructed in E. coli, where the synthetic promoter contained various combinations of phage λ operator regions(O_R2 or O_R3) upstream a weak Plac promoter mutant, while the artificial transcription factor consisted of λ CI as a DNA-binding domain and the C-terminal domain of λ CI fused to N-terminal domain of RNA polymerase α subunit as an effector domain. Various transcriptional outputs were engineered by rationally adjusting a decomposed set of key parameters such as the composition and copy number of the operator upstream the promoter,the presence or absence of the repressor Cro protein, and the temperature sensitivity of wild type CI or its mutant.
Ansari A.Z.,and Mapp A.K.,2002,Modular design of artificial transcription factors,Curr.Opin.Chem.Biol.Biol.,6(6):765-772
Ebright R.H.,and Busby S.,1995,The escherichia Escherichia coli RNA polymerase alpha subunit:Structure and function,Curr.Opin.Gene.Development,5(2):197-203
Hochschild A.,and Dove S.L.,1998,Protein-protein contacts that activate and repress prokaryotic transcription,Cell,92(5):597-600
Hochschild A.,and Lewis M.,2009,The bacteriophage lambda CI protein finds an asymmetric solution,Curr.Opin.Structural Biol.,19(1):79-86
Hochschild A.,Douhan J.,and Ptashne M.,1986,How lambda repressor and lambda Cro distinguish between OR1 and OR3,Cell,47(5):807-816
Joung J.K.,Ramm E.I.,and Pabo C.O.,2000,A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions,Proc.Natl.Acad.Sci.USA,97(13):7382-7387
Khalil A.S.,Lu T.K.,Bashor C.J.,Pyenson N.C.,Joung J.K.,and Collins J.J.,2002,A synthetic biology framework for programming eukaryotic transcription functions,Cell,150(3):647-658
Levine M.,and Manley J.L.,1989,Transcriptional repression of eukaryotic promoters,Cell,59(3):405-408
Li S.C.,Zhu X.D.,Chen Z.L.,and Huang P.T.,2005,Recent progress in study on artificial transcription factors,Junshi Yixue Kexueyuan Yuankan(Academy of Military Medical Sciences),29(5):474-479(李世崇,朱旭东,陈昭烈,黄培堂,2005,人工转录因子的研究进展,军事医学科学院院刊,29(5):474-479)
Pabo C.O.,Sauer R.T.,Sturtevant J.M.,and Ptashne M.,1979,The lambda repressor contains two domains,Proc.Natl.A-cad.Sci.USA,76(4):1608-1612
Ptashne M.,1997,Control of gene transcription:An outline,Nature Med.,3(10):1069-1072
Renkawitz R.,1990,Transcriptional repression in eukaryotes,Trends Genet,6(6):192-196
Sera T.,2009,Zinc-fnger-based artifcial transcription factors and their applications,Adv.Drug Delivery Rev.,61(7-8):513-526
Zhao X.H.,Zhu X.D.,and Huang P.T.,2005,Artificial transcription factors as tools for gene expression manipulation,Shengwu Gongchengxue(Chinese Journal of Biotechnology),21(3):341-347(赵兴卉,朱旭东,黄培堂,2005,人工转录因子研究进展,生物工程学,21(3):341-347)
基本信息:
DOI：10.13417/j.gab.039.002692
中图分类号:Q75
引用信息:
[1]金倩,杨胜利,魏东芝,等.原核细胞中可调控人工转录元件的构建与表征[J].基因组学与应用生物学,2020,39(06):2692-2697.DOI:10.13417/j.gab.039.002692.
基金信息:
国家自然科学基金(31370080)资助
2019-04-17
2019-04-17
2019-04-17