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[1]杨依伟 赵彩芳 吴 波 秦 晗 何明雄**.利用Golden Gate“One-POT”技术组装运动发酵单胞菌转录单元*[J].应用与环境生物学报,2019,25(01):1-10.[doi:10.19675/j.cnki.1006-687x.2018.04043]
 YANG Yiwei,ZHAO Caifang,WU Bo,et al.One-POT assembly of Zymomonas mobilis transcription unit via Golden Gate*[J].Chinese Journal of Applied & Environmental Biology,2019,25(01):1-10.[doi:10.19675/j.cnki.1006-687x.2018.04043]
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利用Golden Gate“One-POT”技术组装运动发酵单胞菌转录单元*()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
25卷
期数:
2019年01期
页码:
1-10
栏目:
研究论文
出版日期:
2019-02-25

文章信息/Info

Title:
One-POT assembly of Zymomonas mobilis transcription unit via Golden Gate*
文章编号:
201804043
作者:
杨依伟1 赵彩芳3 吴 波1 秦 晗1 何明雄12**
1农业部沼气科学研究所,生物质能技术研究中心 成都 610041
2农业部农村可再生能源开发与利用重点实验室 成都 610041
3四川农业大学小麦研究所 成都 611130
Author(s):
YANG Yiwei1 ZHAO Caifang3 WU Bo1 QIN Han1 & HE Mingxiong12**
1 Biomass Energy Technology Research Centre, Biogas Institute of Ministry of Agriculture, Chengdu 610041, China?
2 Key Laboratory of Development and Application of Rural Renewable Energy, Ministry of Agriculture, Chengdu 610041, China?
3 Triticeae Research Institute, Sichuan Agricultural University, Chengdu 611130, China
关键词:
分子组装Golden Gate运动发酵单胞菌转录单元IIS型限制内切酶
Keywords:
molecular assembly Golden Gate Zymomonas mobilis transcription unit type IIS restriction endonuclease
DOI:
10.19675/j.cnki.1006-687x.2018.04043
摘要:
为了研究运动发酵单胞菌合成生物学及代谢工程,和评估运动发酵单胞菌内源启动子及终止子,需要建立一种方便有效的多基因代谢途径组装方法,本研究通过Golden Gate方法构建了一个含有运动发酵单胞菌乙醇脱氢酶2编码基因启动子区(Padh2)及丙酮酸脱羧酶编码基因终止子区(Tpdc)和绿色荧光蛋白开放阅读框(gfp)的人工转录单元。将含有IIS型限制内切酶BbsI识别和酶切序列的特异性接头添加到Padh2、Tpdc和gfp序列两侧,从而保证在一个反应体系中完成上述三个生物元件的酶切和连接,因此该技术被称作“One-POT”。插入上述人工转录单元的穿梭质粒导入大肠杆菌和运动发酵单胞菌后可检测到发出绿色荧光的细胞。本研究结果证实通过Golden Gate方法可有效组装转录单元所需DNA生物元件。本研究不仅为运动发酵单胞菌合成生物学和代谢工程研究,尤其是设计多基因代谢途径组装方面提供了技术工具,也为评估运动发酵单胞菌内源启动子和终止子提供了一种有效方法。(图5表2参34)
Abstract:
In order to study synthetic biology and metabolic engineering in Z. mobilis, besides evaluate native promoters and terminators of Z. mobilis, a convenient and effective multigene metabolic pathway assembly method needs to be established. In this study, an artificial transcriptional unit (TU), which comprises the promoter region of ethanol dehydrogenase 2-encoding gene (Padh2) and the terminator region of pyruvate decarboxylase-encoding gene (Tpdc) of Zymomonas mobilis and open reading frame (ORF) of green fluorescent protein (gfp), were assembled via Golden Gate. Specific adapters containing the sequence of type IIS restriction endonuclease BbsI were flanked the above three biological parts to allow the digestion and assembly of Promoter, ORF and Terminator in one single reaction, thereby being named “One-POT”. The shuttle plasmid embedding the artificial TU was introduced into Escherichia coli and Z. mobilis, and cells with green fluorescence were detected. The results demonstrate that the biological parts of TU were efficiently assembled in the defined order via the Golden Gate method. This study not only provides tools for the research on synthetic biology and metabolic engineering especially on the assembly of multigene metabolic pathway in Z. mobilis, but also offers an effective means to evaluate native promoters and terminators of Z. mobilis.

备注/Memo

备注/Memo:
收稿日期: 2018-04-28 接受日期 Accepted: 2018-06-01
*中国农业科学院基本科研业务费(1610012016020,Y2017LM13,Y2018LM09和Y2018PT81)、国家自然科学基金项目(31570055)和四川省科技计划项目(17ZDYF1305)资助?
**通讯作者(E-mail: hemingxiong@caas.cn)
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更新日期/Last Update: 2018-08-02