Inspired by the Georgia Tech Zinc biosensor, the Lambert High School iGEM Team studied protein degradation to enhance the development of modern biosensors. Today, biosensors are complex biological systems that provide a more reliable and affordable alternative to expensive diagnostic tests. However, in many biosensors, the overexpression of one pigment over another can result in inaccurate readings and subsequently faulty diagnosis. To address this issue, the Lambert iGEM Team devised a “switch” - a genetically engineered construct that degrades SsrA-tagged proteins upon induction by IPTG. Ultimately, our main goal was to quantify the relative strength of degradation and subsequently further characterize a known protease system.

In bacterial cells, SsrA is a monocistronic gene that codes for a specialized strand of RNA known as tmRNA; tmRNA is responsible for rescuing ribosomes from abnormally truncated mRNA templates that lack terminator codons.

This process is the SsrA-tagged degradation pathway, whereby tmRNA binds with the ribosome and cotranslationally adds a degradation tag to the defective mRNA strand, hence marking the nascent polypeptide for degradation. (Tao et. al., 2015) In effect, this degradation tag, called the SsrA tag, serves as protein quality control in the cell by preventing the accumulation of aberrant and incomplete proteins. (Keiler et. al., 1996) These tagged proteins are then transported to some protease by a bacterial adaptor. In E. coli cells, SspB is the bacterial adaptor that transports tagged proteins to the ClpXP protease. Because this pathway is the most common form of proteolysis in prokaryotes, we decided to exploit this pathway to control the overexpression of pigments in biosensors.

ClpXP is classified as an ATP dependent protease (ATPase) that harness energy released from ATP hydrolysis to render protein degradation. Moreover, ClpXP is a protein mechanism that is composed of two separate proteins - ClpX and ClpP. ClpX is the protein that unfolds and translocates the tagged protein into a sequestered proteolytic compartment in ClpP; ClpP is the protein that breaks the individual covalent bonds (polypeptide bonds) that exist between the individual amino acids of the primary structure of the protein.

Accordingly, we devised an inducible genetic construct to study how ClpXP degrades SsrA-tagged GFP and chromoproteins. The data was quantified with the help of a device that could capture the strength of the light reflected by the reporter before, during, and after induction. Ultimately, the main goal of our project was to measure the relative strength of degradation of a tagged reporter and consequently further characterize a protease mechanism.