● Spatial and temporal manipulation of protein abundance is the basis of designing and constructing complicated synthetic regulatory networks (Figure 1)

Figure 1. The mindset of mammalian cell synthetic biology

● Wide variety of synthetic biological tools have been developed to precisely control the protein synthesis on genomic, transcriptional, and translational level.

● Limited tools have been demonstrated to control protein degradation in synthetic biology (Figure 2).

Figure 2. Schematic representation of TPD methods in SynBio (A)Degron (B)PROTACs (C)Trim-Away

● Genetically encoded tool for signal-responsive degradation of untagged proteins is still needed.

This year, we oriented our efforts towards a highly modularized, genetically-encoded target protein degradation directly controlled by exogenous signals to manipulate untagged protein abundance.

To sum up, PREDATOR Pro is tunable, reversible and highly modularized protein degradation tool with promising applications.

A: PREDATOR Pro would be helpful for SynBio researchers to manipulate untagged endogenous protein abundance in a freely selectable temporal and dose-dependent combinations.

B: For scientists working on gene function researches, PREDATOR Pro can also provide solution on developing cellular or animal model to knockdown the expression of specific gene in a tissue specific manner under given stimulus.

C: It's possible that our project might be used for therapeutic purposes. PREDATOR Pro can be used to control the target protein abundance within physical level, thus maintaining the original function of such protein.

Figure 4. Characterization of GFP PREDATOR Pro system based on the DocS-Coh2 interaction.

● The constitutive dimerization of DocS and Coh2 would function as the interface to bring the truncated Trim21 into proximity with GFP protein and trigger the degradation of GFP (Fig 4A).

● ~55% decrease of green fluorescence was observed in pEGFP plasmid and GFP PrePro plasmid co-transfected groups 48 hours after transfection (Fig 4B).

● Western blotting analysis showed that GFP was significantly degraded to ~40% of the control group (Fig 4C), which confirmed that PREDATOR Pro could be used to degrade target protein with high efficiency.

● Significant lower GFP fluorescence could be observed in GFP PrePro expressing group in every time point from 12 hours to 144 hours post transfection. Of note, such trend was also well aligned to the prediction of our mathematical model (Fig 4D and 4E).

Conclusion:

the original PRYSPRY/IgG-Fc interface in the PREDATOR system could be replaced with other protein dimerization pairs while maintaining the protein targeting and degradation activity.

Figure 5. Dual luciferase assay showing the GFP degradation mediated by GFP PrePro system.

● A dual luciferase reporter (pEFR) was used to obtain more accurate GFP abundance changes (Figure 5A), in which firefly luciferase (Fluc) was fused with GFP.

● Results showed significant lower (~50% lower) Fluc/Rluc ratio in GFP PrePro group comparing to the Ctr group (Figure 5B).

Conclusion:

GFP PrePro has impressive efficiency in degrading the target protein

Figure 6. Rapamycin-induced PREDATOR Pro system based on the FRB-FKBP interaction module.

● Version 1.0 of Rapamycin-induced PREDATOR Pro system (RiPrePro1.0 or RiPrePro-1) was constructed by replacing DocS-Coh2 with FRB-FKBP.

● Slight decrease (~20%) of GFP fluorescence in RiPrePro1.0 expressing group under 1 ng/μL rapamycin induction.

How to improve?

● With the input provided by the mathematical model, the parameter reflecting the interaction strength of the protein dimerization pair:

> increasing the concentration of rapamycin

> increasing the binding strength of the interface part of the protein.

Increase Rapamycin concentration:

Figure 7. Fluorescence Heatmap in cells treated with increasing concentrations of Rapamycin.

● The degradation effect could be significantly improved with the increasing concentration of rapamycin.

● Increasing rapamycin would significantly reduce the cellular protein synthesis.

With the implications obtained from the model, we changed the GFPnano-FKBP unit into GFPnano-FKBP-FKBP(Fig 8A).

Figure 8. Rapamycin-induced PREDATOR Pro system based on the FRB-FKBP-FKBP interaction module.

● Dual luciferase assay showed that the normalized GFP abundance in RiPrePro2.0 group was significantly lower than the RiPrePro1.0 group in most time points (Fig 8B), indicating an improved degradation efficiency under increased FKBP copies.

● Further analysis on RiPrePro2.0 further revealed that the GFP degradation activity of RiPrePro2.0 was dose dependent to the rapamycin concentration (Fig 8C).

● Western blotting analysis(Fig 8D) is in alignment with the dual luciferase assay results.

● Reduction on Fluc/Rluc ratio in the RiPrePro2.0 group could be observed in different host cell lines , suggesting good robustness of such RiPrePro system (Fig 8E).

Conclusion:

PREDATOR Pro system can be successfully engineered to degrade the target protein under the control of exogenous signals with decent performance and satisfying robustness.

Figure 13. Representative fluorescence images of HEK-293T cells carrying pEFR and RiPrePro-2 plasmids in comparison to the Ctr group 48 h post 2 ng/μL rapamycin induction.

● The model group discovered that a parameter regarding the protein interaction intensity of FKBP and FRB was pivotal to the degradation efficiency after performing Sensitivity Analysis. Therefore, we designed RiPrePro2.0 composing GFPnano-FKBP*2 and HA-Trim21-FRB.

● Under 2 ng/μL rapamycin induction, the fluorescence intensity of HEK-293T cells co-transfected with pEGFP and RiPrePro2.0 plasmid was significantly lower than the control group.