pTRA – A reporter system for monitoring the intracellular dynamics of gene expression

The presence of standardised tools and methods to measure and represent accurately biological parts and functions is a prerequisite for successful metabolic engineering and crucial to understand and predict the behaviour of synthetic genetic circuits. Many synthetic gene networks are based on transcriptional circuits, thus information on transcriptional and translational activity is important for understanding and fine-tuning the synthetic function. To this end, we have developed a toolkit to analyse systematically the transcriptional and translational activity of a specific synthetic part in vivo. It is based on the plasmid pTRA and allows the assignment of specific transcriptional and translational outputs to the gene(s) of interest (GOI) and to compare different genetic setups. By this, the optimal combination of transcriptional strength and translational activity can be identified. The design is tested in a case study using the gene encoding the fluorescent mCherry protein as GOI. We show the intracellular dynamics of mRNA and protein formation and discuss the potential and shortcomings of the pTRA plasmid.

精准测量与表征生物元件及功能的标准化工具与方法，是实现成功代谢工程的必要前提，同时也是理解、预测合成基因回路行为的核心关键。多数合成基因网络以转录回路为核心构建基础，因此转录与翻译活性相关信息对于理解并精准调控合成功能至关重要。为此，我们开发了一套系统性分析工具套件，可在体内（in vivo）环境中对特定合成元件的转录与翻译活性进行检测。该套件基于质粒pTRA构建，能够将特定转录与翻译输出量精准分配至目标目的基因（gene(s) of interest, GOI），并可对不同遗传构建体系进行横向比较。通过该流程，可筛选得到转录强度与翻译活性的最优组合。本研究以编码荧光mCherry蛋白的基因为目的基因，通过案例研究对该工具套件的设计方案进行了验证。我们展示了mRNA与蛋白质合成的细胞内动态变化过程，并讨论了pTRA质粒的应用潜力与现存局限性。