Kashiwagi Fernanda Miyuki, Wendler Miranda Brenno, de Oliveira Pedrosa Fabio, de Souza Emanuel Maltempi, Müller-Santos Marcelo
Postgraduate Program in Science (Biochemistry), Department of Biochemistry and Molecular Biology, Nitrogen Fixation Laboratory, Federal University of Paraná (UFPR), Curitiba, Brazil.
Biological Sciences Undergraduate Course, Department of Biochemistry and Molecular Biology, Nitrogen Fixation Laboratory, Federal University of Paraná (UFPR), Curitiba, Brazil.
Front Bioeng Biotechnol. 2021 Sep 16;9:730967. doi: 10.3389/fbioe.2021.730967. eCollection 2021.
Control of gene expression is crucial for several biotechnological applications, especially for implementing predictable and controllable genetic circuits. Such circuits are often implemented with a transcriptional regulator activated by a specific signal. These regulators should work independently of the host machinery, with low gratuitous induction or crosstalk with host components. Moreover, the signal should also be orthogonal, recognized only by the regulator with minimal interference with the host operation. In this context, transcriptional regulators activated by plant metabolites as flavonoids emerge as candidates to control gene expression in bacteria. However, engineering novel circuits requires the characterization of the genetic parts (e.g., genes, promoters, ribosome binding sites, and terminators) in the host of interest. Therefore, we decomposed the QdoR regulatory system of , responsive to the flavonoid quercetin, and reassembled its parts into genetic circuits programmed to have different levels of gene expression and noise dependent on the concentration of quercetin. We showed that only one of the promoters regulated by QdoR worked well in , enabling the construction of other circuits induced by quercetin. The QdoR expression was modulated with constitutive promoters of different transcriptional strengths, leading to low expression levels when QdoR was highly expressed and vice versa. strains expressing high and low levels of QdoR were mixed and induced with the same quercetin concentration, resulting in two stable populations expressing different levels of their gene reporters. Besides, we demonstrated that the level of QdoR repression generated different noise levels in gene expression dependent on the concentration of quercetin. The circuits presented here can be exploited in applications requiring adjustment of gene expression and noise using a highly available and natural inducer as quercetin.
基因表达的控制对于多种生物技术应用至关重要，特别是对于实现可预测和可控的遗传回路而言。此类回路通常通过由特定信号激活的转录调节因子来实现。这些调节因子应独立于宿主机制发挥作用，具有低的本底诱导或与宿主成分的串扰。此外，信号也应是正交的，仅被调节因子识别，对宿主操作的干扰最小。在这种情况下，由植物代谢物如黄酮类化合物激活的转录调节因子成为控制细菌基因表达的候选者。然而，构建新的回路需要对目标宿主中的遗传元件（如基因、启动子、核糖体结合位点和终止子）进行表征。因此，我们分解了对黄酮类化合物槲皮素响应的 的QdoR调节系统，并将其元件重新组装成遗传回路，这些回路根据槲皮素的浓度被编程为具有不同水平的基因表达和噪声。我们表明，由QdoR调节的启动子中只有一个在 中运行良好，从而能够构建由槲皮素诱导的其他回路。QdoR的表达通过不同转录强度的组成型启动子进行调节，当QdoR高表达时导致低表达水平，反之亦然。将表达高水平和低水平QdoR的 菌株混合并用相同浓度的槲皮素诱导，产生了两个稳定的群体，它们表达不同水平的基因报告物。此外，我们证明了QdoR的抑制水平根据槲皮素的浓度在基因表达中产生不同的噪声水平。本文展示的回路可用于需要使用高度可用的天然诱导剂槲皮素来调节基因表达和噪声的应用中。
