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《工程(英文)》 >> 2018年 第4卷 第6期 doi: 10.1016/j.eng.2018.09.012

一种使用隔离子RiboJ测量绝对定量蛋白表达量的方法

a West China Medical Center Team for International Genetically Engineered Machine Competition 2017, West China School of Medicine / West China Hospital, Sichuan University, Chengdu 610041, China

b Department of Gastroenterology, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu 610041, China

c West China School of Stomatology, West China Hospital, Sichuan University, Chengdu 610041, China

d College of Physical Science and Technology, Sichuan University, Chengdu 610064, China # These authors contributed equally to this work

收稿日期: 2018-03-07 修回日期: 2018-04-20 录用日期: 2018-09-29 发布日期: 2018-10-09

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摘要

特定启动子的绝对定量蛋白表达量测定在分子生物学及合成生物学领域具有重要的意义。启动子强度一般由该启动子表达荧光蛋白的荧光强度表示。然而,对于特定启动子的绝对定量蛋白表达量测定方法目前仍不尽如人意。不同蛋白编码序列对启动子表达水平的影响及现有测定绝对定量蛋白表达量方法的复杂性,均为绝对定量蛋白表达量测定带来了挑战。我们在这里介绍了一种利用隔离子RiboJ和标准荧光曲线快速测定绝对定量蛋白表达量的方法,并通过模型对该方法进行了验证。使用这种方法,我们成功测定了Anderson启动子家族中9种组成型启动子的强度。本方法为代谢通路设计提供了更准确的数据,并为不同启动子强度的标准化提供了一种直接的方案。

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参考文献

[ 1 ] Maeda M, Shimada T, Ishihama A. Strength and regulation of seven rRNA promoters in Escherichia coli. PLoS ONE 2015;10(12):e0144697. 链接1

[ 2 ] Rudge TJ, Brown JR, Federici F, Dalchau N, Phillips A, Ajioka JW, et al. Characterization of intrinsic properties of promoters. ACS Synth Biol 2016;5 (1):89–98. 链接1

[ 3 ] Pedraza JM, Van Oudenaarden A. Noise propagation in gene networks. Science 2005;307(5717):1965–9. 链接1

[ 4 ] Chappell J, Freemont P. In vivo and in vitro characterization of r70 constitutive promoters by real-time PCR and fluorescent measurements. In: Polizzi KM, Kontoravdi C, editors. Synthetic biology, 1073. Totowa: Humana Press; 2013. p. 61–74. 链接1

[ 5 ] Kelly JR, Rubin AJ, Davis JH, Ajo-Franklin CM, Cumbers J, Czar MJ, et al. Measuring the activity of BioBrick promoters using an in vivo reference standard. J Biol Eng 2009;3(1):4. 链接1

[ 6 ] Luo Y, Zhang L, Barton KW, Zhao H. Systematic identification of a panel of strong constitutive promoters from Streptomyces albus. ACS Synth Biol 2015;4(9):1001–10. 链接1

[ 7 ] LouC,StantonB,ChenY,MunskyB,VoigtCA.Ribozyme-basedinsulatorpartsbuffer synthetic circuits from genetic context. Nat Biotechnol 2012;30(11):1137–42.

[ 8 ] Anderson JC. Anderson promoter collection [Internet]. 2018 [cited 2018 Mar 6]. Available from: http://parts.igem.org/Promoters/Catalog/Anderson.

[ 9 ] Beal J, Haddock-Angelli T, Gershater M, De Mora K,Lizarazo M, Hollenhorst J, et al. Reproducibility of fluorescent expression from engineered biological constructs in E. coli. PLOS One 2016;11(3):e0150182. 链接1

[10] Song K. Introduction to synthetic biology. Beijing: Science Press; 2010. Chinese.

[11] Deft TU. Modeling [Internet]. 2018 [cited 2018 Mar 6]. Available from: http:// 2015.igem.org/Team:TU_Delft/Modeling.

[12] Agilent. E. coli cell culture concentration from OD600 calculator [Internet]. 2018 [cited 2018 Mar 6]. Available from: https://www.genomics.agilent.com/ biocalculators/calcODBacterial.jsp.

[13] Berg JM, Tymoczko JL, Stryer L, editors. Biochemistry: a short course. New York: W. H. Freeman and Company; 2012.

[14] Anderson JC. Strength of Anderson Promoter [Internet]. 2018 [cited 2018 Mar 6]. Available from: http://parts.igem.org/wiki/index.php/Part:BBa_ J23100.

[15] William and Mary Team. RiboJ [Internet]. 2018 [cited 2018 Apr 19]. Available from: http://2016.igem.org/Team:William_and_Mary/RiboJ.

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