Team:Lambert GA/Design
Design
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 pλR).
Without IPTG induction, pλR 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 pλR 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: pλR LacI tsPurple pLac ClpXP CI, pλR LacI tsPurpleDAS pLac ClpXP CI, and pλR 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.
Construct Design
