Abstract

  A major problem in modern biology is a lack of well understood microbial sensors and one limitation in metabolic engineering is an inability to control dynamic cell metabolism. Our project provides a solution to both problems with synthetic biology via a synthetic sensing scaffold. There are no standardized platforms for sensor characterization today, and as synthetic biology grows, so does the need to develop standards for the creation of genetic construct. The completion of this project provides a high throughput method of developing a library of microbial sensors, a mechanism for controlling cell metabolism based on environmental conditions, and a standardized platform for the design and characterization of synthetic sensing circuits.

     This project aims to introduce a “plug-and-play” method for sensing circuits, and to provide a protocol for characterizing novel sensors. A two-plasmid system, with a sensor and reporter, is employed in our standard. The sensor is created by generating a chimera between a sensing gene and a histidine kinase, which activates a transcriptional regulation protein. This protein controls the activity of the reporter. This upregulated reporter with a fluorescent protein will respond by fluorescing in the presence of our target due to sensor activity.

   A wide range of sensing domains can be used and characterized in this manner, making this system highly modular. This modularity leads to high throughput sensor characterization, which can be used with both known and unknown sensing domains. A successful biosensor circuit platform helps bridge the gap in today’s protocols, modeling the work that should be done in all aspects of the field of synthetic biology, not only in biosensor construction.