PDF(Pubmed)
Abstract：
Cyanobacteria utilize sunlight to convert carbon dioxide into a wide variety of secondary metabolites and show great potential for green biotechnology applications. Although cyanobacterial synthetic biology is less mature than for other heterotrophic model organisms, there are now a range of molecular tools available to modulate and control gene expression. One area of gene regulation that still lags behind other model organisms is the modulation of gene transcription, particularly transcription termination. A vast number of intrinsic transcription terminators are now available in heterotrophs, but only a small number have been investigated in cyanobacteria. As artificial gene expression systems become larger and more complex, with short stretches of DNA harboring strong promoters and multiple gene expression cassettes, the need to stop transcription efficiently and insulate downstream regions from unwanted interference is becoming more important. In this study, we adapted a dual reporter tool for use with the CyanoGate MoClo Assembly system that can quantify and compare the efficiency of terminator sequences within and between different species. We characterized 34 intrinsic terminators in Escherichia coli, Synechocystis sp. PCC 6803, and Synechococcus elongatus UTEX 2973 and observed significant differences in termination efficiencies. However, we also identified five terminators with termination efficiencies of >96% in all three species, indicating that some terminators can behave consistently in both heterotrophic species and cyanobacteria.
摘要：
蓝藻利用阳光将二氧化碳转化为多种次生代谢产物，并显示出绿色生物技术应用的巨大潜力。尽管蓝细菌合成生物学不如其他异养模式生物成熟，现在有一系列分子工具可用于调节和控制基因表达。基因调控的一个领域仍然落后于其他模式生物是基因转录的调节，特别是转录终止。大量内在转录终止子现在在异养生物中可用，但是在蓝藻中只研究了一小部分。随着人工基因表达系统变得越来越大、越来越复杂,带有强启动子和多个基因表达盒的短DNA片段，有效地停止转录并使下游区域免受不必要的干扰的需要变得越来越重要。在这项研究中,我们采用了一种双报告工具，用于CyanoGateMoClo组装系统，该系统可以量化和比较不同物种内部和之间的终止子序列的效率。我们表征了大肠杆菌中的34种内在终止子，集胞藻。PCC6803和延伸神经球菌UTEX2973，并观察到终止效率的显着差异。然而,我们还确定了在所有三个物种中终止效率>96％的五个终止子，表明某些终止子在异养物种和蓝细菌中的行为一致。
