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“One-POT” assembly of Zymomonas mobilis transcription unit via Golden Gate(PDF)

Chinese Journal of Applied & Environmental Biology[ISSN:1006-687X/CN:51-1482/Q]

2019 01
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“One-POT” assembly of Zymomonas mobilis transcription unit via Golden Gate
YANG Yiwei1 ZHAO Caifang3 WU Bo1 QIN Han1 & HE Mingxiong1 2**
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
molecular assembly Golden Gate Zymomonas mobilis transcription unit type IIS restriction endonuclease
Q78: Q939.9

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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Last Update: 2019-02-25