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.For example, in the hypothetical biosensor X, the system is designed such that it will express purple when the concentration of X is less than 4𝞵M, orange when the concentration of X is between 4𝞵M and 6𝞵M, and red when the concentration of X is between 6𝞵M and 10𝞵M. However, due to leakiness that may exist in the system or fluctuations in the affinities of the biosensing promoters, the purple reporter is overexpressed, as evident by the figure the below.

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 adapter 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 harnesses 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. We grew these constructs in DH10 E. coli strains; however, since the DH10 strains have naturally occurring ClpXP, we decided to grow these constructs in Keio Wild knockout strains to accurately quantify the relative strength of degradation. We were able to successfully grow our constructs in the Keio ClpP Knockout strains and one of the constructs in the Keio Wild strain, but due to the time constraints, we were unable to gain any results with the ClpX Knockout strain. We used the fluorescent plate reader at the Styczynski Lab to obtain these results. 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.

Improving Previous Parts

Our 2016 iGEM team used existing Biobrick parts from the Uppsala team (BBa_K1033906) and also the Endy Lab (BBa_M0050-LAA, M0052-DAS). We furthered Edinburgh’s work by expanding upon their experiments with GFP and the degradation tags, replacing GFP with the tsPurple chromoprotein and characterizing the ClpXP system.

We first obtained the tsPurple sequence from the 2013 Uppsala iGEM wiki page. The sequence was ordered online from IDT, hydrated, and assembled with 3A assembly to create our constructs. We obtained the tsPurple with no degradation tag, tsPurple with the LAA degradation tag, and tsPurple with the DAS degradation tag. The degradation tags were obtained from the Endy Lab sequencing and correspond to strong degradation for LAA and moderate for DAS.

We sought to improve upon the LAA and DAS degradation tags by demonstrating their relative strength in degrading the tsPurple chromoprotein and to improve upon the characterization of the tsPurple gene so that its relative degradation may be present for anyone who needs it.


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