School of Chemistry and Chemical Engineering, Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, Shihezi University, Shihezi, 832003, P. R. China.
Chembiochem. 2019 Sep 16;20(18):2383-2389. doi: 10.1002/cbic.201900163. Epub 2019 Jul 8.
The design of improved synthetic components is an important research field in synthetic biology. The terminator, responsible for terminating gene transcription, is a necessary component for yeast gene expression. The efficiency element, the positioning element and the poly(A) site have been identified as the constituent parts necessary for the yeast terminator to perform its function. However, the functions of linker 1 (situated between the efficiency element and the positioning element) and linker 2 [between the positioning element and the poly(A) site] in the terminator are still controversial. Here, we have thus designed and synthesized a yeast synthetic terminator library incorporating random 10 bp linker 1 units. For indirect characterization of the strengths of 266 synthetic terminators with the aid of the enhanced green fluorescent protein (eGFP), their fluorescence intensity (FI) values were determined; they ranged from 2.3648 to 3.5270, thus indicating that the strength of yeast terminator can be finely adjusted by changing the linker 1 sequence. The strength increased with decreasing GC content in linker 1, with a T-rich linker 1 helping to enhance terminator strength further. Reducing the stem length can increase the gene expression in cases of weak and medium-strength terminators but decreases the gene expression of strong terminators. Deletion of linker 2 seems to have a positive effect on weak and medium-strength terminators. Construction of a lycopene biosynthesis pathway with synthetic terminators effectively regulated lycopene synthesis, thus indicating that it is highly feasible to use terminators for fine regulation of gene and pathway expression.
改进合成元件的设计是合成生物学的一个重要研究领域。终止子负责终止基因转录，是酵母基因表达所必需的组成部分。效率元件、定位元件和多聚腺苷酸（poly(A)）位点已被确定为酵母终止子发挥功能所必需的组成部分。然而，终止子中连接子 1（位于效率元件和定位元件之间）和连接子 2[位于定位元件和 poly(A) 位点之间]的功能仍存在争议。在这里，我们设计并合成了一个包含随机 10bp 连接子 1 单元的酵母合成终止子文库。为了借助增强型绿色荧光蛋白（eGFP）间接表征 266 个合成终止子的强度，我们测定了它们的荧光强度（FI）值；FI 值范围为 2.3648 至 3.5270，这表明通过改变连接子 1 序列可以精细调节酵母终止子的强度。连接子 1 中的 GC 含量越低，强度越大，富含 T 的连接子 1 有助于进一步增强终止子的强度。降低茎的长度可以增加弱和中等强度终止子的基因表达，但会降低强终止子的基因表达。删除连接子 2 似乎对弱和中等强度终止子有积极影响。用合成终止子构建的番茄红素生物合成途径有效地调节了番茄红素的合成，这表明使用终止子精细调节基因和途径表达是非常可行的。
