Engineering leave-one-out GFP based biosensors

Abstract

To this end we proposed the development of a biosensor platform based on the green fluorescent protein (GFP) called leave-one-out GFP (LOO-GFP) biosensors. This platform is built on the principle that when one of the 11 β-strands in GFP is excised (LOO-GFP) there is a loss in the native fluorescence of the molecule that can be reconstituted upon the addition of the missing peptide. Additionally, the pocket created as a result of the omission of any of the β-strands can be redesigned to accommodate an exogenous peptide sequence, allowing for LOO-GFPs to function as designable biosensors. Since this approach is a redesign of the existing molecule, we conducted a mechanistic study of the structure function relationship in GFP so as to identify the best engineering strategies. This work focuses on how the production of the fluorescent para-hydroxbenzylidene imidazolinone chromophore is regulated, the identification of optimal design scaffolds and developing methods that can be used to design LOO-GFP biosensors for diagnostics and environment surveillance.

Biosensors are commonly employed in a plethora of fields that include drug discovery, medical diagnostics, environmental surveillance, quality control and the detection of biological warfare agents. This broad spectrum of uses is generally achieved by varying the bio-recognition elements in these devices. The choice of selecting antibodies, enzymes, cells or nucleic acids as a bio-receptor of choice is particularly important since it will dictate how the signal associated with the detection of a given analyte is transduced and ultimately outputted. Coupling of these elements within biosensors allows for the development of robust devices with high detection sensitivity and specificity. These devices however, are not always portable or cheap enough to manufacture at a large scale. As a result, they can only be used sparingly in economically deprived regions and in areas where there is a threat of a pandemic or biological warfare agents. Such limitations underscore the need for the development of newer classes of biosensors capable of rapid detection with high sensitivity and specificity.