A New Environmental Biosensor for Cell Free Synthetic Biological Systems

Abstract

Biosensors utilize fundamental properties of biophysics to enable detection of environmental analytes. While these systems are frequently based on living cells, they have potential applications in multiple fields. Cell-free synthetic biological systems, such as artificial cells (i.e., liposomal encapsulations of functional biological parts), are an example of systems that could be enhanced by new biosensors. Here, we created a biosensor based on the biophysical interactions of biotin and streptavidin. Biotin plays an essential role in cell growth and the typical amount of biotin required by cells is low (e.g., 1 ng/ml in E. coli). Biotin is also widely used in molecular assembly because of its strong conjugation to streptavidin, with a Kd around 10−15 M). We leveraged this strong attraction in a competitive binding scheme to create a biotin sensor that is both specific and sensitive in comparison to common biotin assay methods that are based on radioactive labeling, microbiological, or physicochemical principles. This new biosensor has a detection limit in pg range, and significantly discriminates between biotin and its metabolic precursor in E. coli, dethiobiotin. This engineered biosensor can be used as a biotin detector for biotin synthesis by engineered cells. Additionally, it can be deployed in cell-free synthetic systems. Ultimately, our engineered biosensor can be coupled to cell-free systems to act as an environmental reporter. Alternatively, it can trigger cell-free gene expression in artificial cells. We anticipate this new technology will impact work in fields ranging from synthetic biology to the biophysics of biomaterial assembly.Acknowledgements: We gratefully acknowledge funding from the Air Force Office of Scientific Research.