Engineering an inducible NO pathway to facilitate cell-electronics communication

Abstract

Turning cells into useful devices to perform unnatural functions creates the potential to permit the interface between biological organisms and electronics. In this thesis cell-based devices were designed and constructed to respond to either light or a specific chemical stimulus. Design constraints were defined by the eventual application of the device, a biohybrid robot. The enzyme endothelial nitric oxide synthase (eNOS) was chosen as a target for genetic engineering. Prior to constructing the device a suitable host for the engineered construct was selected. CHO-K1 cells were transfected with nitric oxide synthase and expression levels were characterized via flow cytometry and inhibitor studies. A novel method for the effective delivery of inhibitors was developed and applied to demonstrate that transfected eNOS was sufficiently expressed to produce a measurable output. In addition, a balance between the native nitric oxide production machinery of the cells and the transfected endothelial nitric oxide synthase was observed. Two systems were designed and constructed for stimuli responsive nitric oxide production. The first system was designed to produce nitric oxide in response to the presence of the antibiotic rapamycin. Chemical induced dimerization would bring the two separated domains of endothelial nitric oxide synthase into close enough proximity to re-establish protein function. The separate oxygenase and reductase domains were successfully amplified and subsequently fused with components of the chemically induced dimerization system. The second system involved fusing a domain from the plant gene Nhp1 (Light Oxygen Voltage domain - LOV) capable of harvesting a photon, with mouse endothelial nitric oxide synthase. This strategy aimed to hijack the wild type protein’s native electron transfer pathway. Manipulation was carried out in bacteria with subsequent transfection into CHO-K1 cells. Subsequent testing of nitric oxide production the mutant cells confirmed the optical sensitivity of the mutant eNOS. Moreover both LOV mutant cell lines were capable of fast response times and switching behaviour. The findings of this thesis demonstrate that genetic engineering of endothelial nitric oxide synthase is a suitable strategy for the controlled release of nitric oxide upon optical stimulation. Moreover the potential of an engineered cell to respond quickly to stimuli has been realized, comparing favourably to genetically engineered systems that rely on gene expression to elicit an output.