To further characterize ClpXP, we created an inducible genetic circuit in which ClpXP will degrade the desired protein. We designed two reporter systems with 3 levels of degradation tags.
Before induction, the LacI gene will produce lac inhibitor molecules that will bind to the pR11 (pLac1) site and inhibit it from promoting ClpXP and the CI. This system allows tsPurple to be significantly expressed in the E. coli cells. Upon induction of IPTG, IPTG molecules will bind to the LacI repressor, which thereby allows pR11 to promote ClpXP and the CI. The CI will repress the p-lambda-r promoter to ensure that no additional Lac molecules are being produced. Once expressed, ClpXP will proceed to degrade the chromoprotein, tsPurple.
With this in mind, we assembled six constructs - three with the GFP chromoprotein and three with tsPurple. The three GFP constructs served as our baseline constructs as we can compare the success of failure of the tsPurple constructs; the built GFP constructs work to ensure that our circuit can feasibly deliver results. In each set - the separate GFP and tsPurple constructs- the first construct serves as our control due to the lack of degradation tags on the reporters. In the second construct, the reporters are associated with the DAS degron; and in the third construct, the reporters were tagged with the LAA degron. According to the biobrick registry, LAA, is a much stronger degradation tag than DAS; consequently, we expect to see a greater degree of degradation (significantly lighter color of purple) in the constructs with the LAA tag than the constructs with the DAS tag and no degradation tags.
Moreover, we have assembled constructs in two different knockout strains:
- Keio ClpX Knockout: In this strain, ClpX will be knocked out and ClpP will be allowed to function. ClpX is responsible for de-linearizing the target protein into its primary structure and then translocating it into a proteolytic cavity in ClpP. Since ClpX will not be expressed, the chromoprotein will not be de-linearized and hence prevent ClpP from degrading the primary structure of the protein. With this in mind, we expect our plates to remain purple in the Keio ClpX Knockout strain.
- Keio ClpP Knockout: In this strain, ClpP will be knocked out and ClpX will be allowed to function. ClpP is responsible for degrading the primary structure of the protein itself; it does this by breaking the individual covalent bonds (polypeptide bonds) that exist between the amino acids in the polypeptide chain. Since ClpP will not be expressed, the chromoprotein will not be degraded into individual amino acids. However, despite this, the chromoprotein will still be degraded partially because it will be de-linearized by ClpX. In other words, although the protein will still be in its primary structure, it will still lack the hydrogen bonds, hydrophobic interactions, ionic bonds, disulfide bridges, and R-Group interactions that exist in its tertiary (or in some cases, quaternary) structure; consequently, that will limit the chromoprotein’s ability to express its pigments. With this in mind, we expect our plates to have a smaller number of purple cells present in the Keio ClpP Knockout strain.