The full genetic construct is comprised of three parts: pλr LacI, one of the three tsPurple parts (tsPurple with no deg tag, tsPurpleDAS, or tsPurpleLAA), and pLac-ClpXP-CI (a LacI repressible promoter, a proteolysis mechanism, and CI to regulate p-lambda-r).

The animation shows the function of the full construct in steps:
1. p-lambda-r expresses LacI, which produces lac inhibitor proteins to repress pLac, and tsPurple chromoproteins
2. IPTG is introduced into the cell and binds with the lac inhibitor proteins, preventing repression of pLac and beginning expression of ClpXP and CI
3. CI prevents any further expression of LacI and tsPurple, while ClpXP recognizes the degradation tag and breaks down tsPurple.

Without IPTG induction, p-lambda-r is naturally expressing and will promote the LacI gene and the tsPurple. The LacI gene produces lac inhibitor proteins that bind to the pLac1 site and inhibit pLac from promoting ClpXP and the CI. This system allows tsPurple to be significantly expressed without any degradation in the E. coli cells. Upon induction of IPTG, IPTG molecules will bind to the LacI repressor, which prevents the lac inhibitor proteins from repressing pLac and allows for expression of ClpXP and the CI. Once expressed, the CI will repress the p-lambda-r promoter to ensure that no additional lac inhibitor proteins (or tsPurple chromoproteins) are being produced. Simultaneously, ClpXP will proceed to degrade the chromoprotein tsPurple when a degradation tag (in our system, either DAS or LAA) is attached.

Ideally, the final result would be three fully assembled constructs: p-lambda-r-LacI-tsPurple-pLac-ClpXP-CI, p-lambda-r-LacI-tsPurpleDAS-pLac-ClpXP-CI, and p-lambda-r-LacI-tsPurpleLAA-pLac-ClpXP-CI. Once all three constructs were induced with IPTG, the relative levels of degradation would be compared; theoretically the construct with no degradation tag would show no degradation, the DAS tag (which is a moderately fast degradation tag) would show a middle level of degradation, and the LAA tag (a fast degradation tag) would show the highest levels of degradation. The Chrome-Q system would then be utilized to measure the HSV values of the three different levels of degradation.

Keio Competent Cells

Keio ClpX Knockout: To prove our genetic construct, Lambert iGEM successfully made competent cells of Keio Wild and Keio ClpP Knock-Out E. Coli from a dehydrated disk donated to the team by the E. Coli Genetic Stock Center. These strains naturally have the ClpXP gene which is a two part mechanism that degrades proteins. ClpX linearizes the protein, then ClpP breaks it down into individual amino acids. Theoretically, once the full genetic construct is inserted in the cell, the Keio Wild strand would have very little expression of the Ts-Purple due to the actions of the ClpXP mechanism. In contrast, the Keio ClpP Knock-out is expected to produce a moderate amount of color because ClpP is not in the cell to finish the degradation process. To add, Keio ClpX Knock-Out would have the most color expression since ClpX is not present to linearize the protein. Thus, ClpP is not able to separate the protein into amino acids, and no degradation would occur. If the results reflected the expected outcome, the genetic construct would be proven correct. However, Lambert iGEM was only able to insert part of the genetic construct, a promoter and Ts-Purple, into the strands and did not receive the expected outcome due to errors in the transformation protocol.