[1]杨依伟,赵彩芳,吴波,等.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):170-175.[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):170-175.[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
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):170-175.[doi:10.19675/j.cnki.1006-687x.2018.04043]
《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:
- 170-175
- 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
- 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
- 分类号:
- Q78: Q939.9
- 摘要:
- 为研究运动发酵单胞菌合成生物学及代谢工程，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. 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）、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3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. 结果显示通过Golden Gate方法可有效组装转录单元所需DNA生物元件. 本研究不仅可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技术工具，也可为评估运动发酵单胞菌内源启动子和终止子提供一种有效方法. （图5 表2 参34）
- Abstract:
- In order to study synthetic biology and metabolic engineering in Zymomonas mobilis, besides evaluating its native promoters and terminators, a convenient and effective multigene metabolic pathway assembly method needs to be established. In this study, an artificial transcriptional unit (TU), which comprised the promoter region of ethanol dehydrogenase 2-encoding gene (Padh2) and the terminator region of pyruvate decarboxylase-encoding gene (Tpdc) of Z. mobilis and an open reading frame (ORF) of green fluorescent protein (gfp), was assembled via the Golden Gate method. Specific adapters containing the sequence of type IIS restriction endonuclease BbsI flanked the above three biological parts to allow the digestion and assembly of the Promoter, ORF, and Terminator in one single reaction, hence the names “One-POT”. The shuttle plasmid embedding the artificial TU was introduced into Escherichia coli and Z. mobilis, and the 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.
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