Svoboda Jessee, Cisneros Brenda, Philmus Benjamin
College of Chemical, Biological, and Environmental Engineering, Oregon State University, Corvallis, OR, USA.
Department of Pharmaceutical Sciences, Oregon State University, Corvallis, OR, USA.
Synth Biol (Oxf). 2021 Sep 1;6(1):ysab019. doi: 10.1093/synbio/ysab019. eCollection 2021.
Cyanobacteria are promising chassis for synthetic biology applications due to the fact that they are photosynthetic organisms capable of growing in simple, inexpensive media. Given their slower growth rate than other model organisms such as and , there are fewer synthetic biology tools and promoters available for use in model cyanobacteria. Here, we compared a small library of promoter-riboswitch constructs for synthetic biology applications in sp. PCC 7120, a model filamentous cyanobacterium. These constructs were designed from six cyanobacterial promoters of various strengths, each paired with one of two theophylline-responsive riboswitches. The promoter-riboswitch pairs were cloned upstream of a chloramphenicol acetyltransferase () gene, and CAT activity was quantified using an assay. Addition of theophylline to cultures increased the CAT activity in almost all cases, allowing inducible protein production with natively constitutive promoters. We found that riboswitch F tended to have a lower induced and uninduced production compared to riboswitch E for the weak and medium promoters, although the difference was larger for the uninduced production, in accord with previous research. The strong promoters yielded a higher baseline CAT activity than medium strength and weak promoters. In addition, we observed no appreciable difference between CAT activity measured from strong promoters cultured in uninduced and induced conditions. The results of this study add to the genetic toolbox for cyanobacteria and allow future natural product and synthetic biology researchers to choose a construct that fits their needs.
蓝细菌是合成生物学应用中很有前景的底盘生物，因为它们是能够在简单、廉价培养基中生长的光合生物。鉴于它们的生长速度比其他模式生物（如 和 ）慢，可用于模式蓝细菌的合成生物学工具和启动子较少。在这里，我们比较了一个小型启动子-核糖开关构建体文库，用于丝状模式蓝细菌集胞藻PCC 7120中的合成生物学应用。这些构建体由六个不同强度的蓝细菌启动子设计而成，每个启动子与两种茶碱响应核糖开关之一配对。启动子-核糖开关对被克隆到氯霉素乙酰转移酶（）基因的上游，并使用 测定法对CAT活性进行定量。在几乎所有情况下，向培养物中添加茶碱都会增加CAT活性，从而允许使用天然组成型启动子进行诱导型蛋白质生产。我们发现，对于弱启动子和中等强度启动子，与核糖开关E相比，核糖开关F的诱导型和非诱导型产量往往较低，尽管非诱导型产量的差异更大，这与之前的研究一致。强启动子产生的基线CAT活性高于中等强度和弱启动子。此外，我们观察到在未诱导和诱导条件下培养的强启动子所测得的CAT活性之间没有明显差异。这项研究的结果丰富了蓝细菌的遗传工具箱，并使未来的天然产物和合成生物学研究人员能够选择适合其需求的构建体。
