Team:BostonU/Demonstrate

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Orthogonality of LOV2 & PhiReX
In order to significantly improve transcriptional control, we aimed to demonstrate that our blue- and red-light inducible systems could be used in conjunction to multiplex control. In order to do so, we needed to demonstrate that their activation spectra were indeed orthogonal.
Absorbance spectra for LOV2 and PhiReX Figure 1: Absorbance spectra of LOV2 and PhiReX. LOV2 peaks at 450-490 nm and PhiReX peaks at 640-670 nm.
Since the peak absorbance wavelengths of PhiReX and LOV2 lie on opposite ends of the visible spectrum, the systems are orthogonal in theory. To confirm this hypothesis, we tested PhiReX and LOV2 orthogonality with both shaker experiments and eVOLVER experiments.
orthogonality dataFigure 2: Average mRuby expression in au of PhiReX and LOV2 under different experimental conditions.
The data above confirms that LOV2 is not activated by red light, and PhiReX is not activated by blue light. Thus, the systems are indeed orthogonal and could be used to implement several layers of control over gene expression.
blue orthogonality dataFigure 3: mRuby expression under PhiReX and LOV2 in a blue light eVOLVER experiment for orthogonality, as well as fold change from negative control strain.
Examining the fold change from negative control for each system in orthogonality experiments, we found that LOV2-driven mRuby expression changes significantly under blue light while PhiReX-driven expression does not. Similarly, PhiReX-driven mRuby expression changes significantly under red light while LOV2-driven expression does not. Although PhiReX expression is higher than negative control in the above figure, this is likely due to either transient activation of PhiReX by ambient white light in the lab, or due to a basal "leakiness" in the promoter system.