Abstract
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 B. subtilis, 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 E. coli, 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. E. coli 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.
Highlights
Cells naturally sense and react to extracellular signals
Bacillus subtilis has the TetR-type negative regulator QdoR that is induced by flavonoids such as quercetin and fisetin (Hirooka et al, 2007)
We reassembled them with synthetic parts and cloned them in plasmids to transform E. coli
Summary
Cells naturally sense and react to extracellular signals This environmental computation is carried out by transcriptional regulators that increase or decrease gene expression upon the appearance of a new molecule in the extracellular medium (or a variation of its concentration). Some soil bacteria naturally recognize metabolites produced and emitted by plants in their root exudates. Bacillus subtilis has the TetR-type negative regulator QdoR that is induced by flavonoids such as quercetin and fisetin (Hirooka et al, 2007). Quercetin inhibits the binding of QdoR to DNA; the transcription of qdoI and qdoR is induced (Hirooka et al, 2007)
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