Team:NUDT CHINA/Engineering

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ENGINEERING SUCCESS

Learn and Improve

 Archives

• Introduction


• Brain storming and literature research
• Modularization and Design
• Test of replaceability of interface
• Learn and Improve

Introduction

In our iGEM 2020 project, tremendous amount of engineering effort has been made to bring the signal responsive feature into the PREDATOR system we kept working on for years (Figure 1). As the most important foundation of the PREDATOR Pro system, the successful reengineer of the Trim21 protein is considered as the most important achievement we made this year. Here in this Engineering Success page, we will discuss how Trim21 protein was engineered to reach our needs and demonstrate the functional validation data of our new part BBa_K3396009, also termed as GFP PrePro, showing that it worked as we expected.

 

 

Figure 1.Engineering flow chart of Predator Pro system

 

Brain storming and literature research

In 2018 and 2019, we have demonstrated the PREDATOR system, an effective endogenous protein degradation system that allows for direct regulation of target protein degradation. Although we have demonstrated that the PREDATOR system can be controlled by different signal responding promoters, thus provided a controllable protein degradation method, we also noticed that such transcriptionally-controlled feature maintains improvable to further decrease the response time lag and improve robustness.

Achieving direct signal responsiveness requires a proper interface to rewire the signal induced protein-protein interaction or other biochemical reactions into the PREDATOR system. Hence, we reanalyzed our previous working model. In the previous PREDATOR system, the nanobody-IgG Fc fusion protein links target protein and Trim21 protein via two important protein-protein interactions. Among them, the nanobody-target interaction determines the specificity of the system, thus remains untouchable. On the other hand, the second key interaction occurs between the IgG Fc domain and the Trim21 protein, seems replaceable. Further analysis on the structure and working mechanism of Trim21 also showed that Trim21 interacts with IgG Fc domain via PRYSPRY domain located on its carboxy terminus.

Modularization and Design

Building on our literature research, we determined to try replacing the Trim21 PRYSPRY domain and the IgG Fc domain linked with the nanobody into other protein dimerization pairs, either constitutive or signal-regulated, to create an interface that direct controls of the protein degradation. Since the nanobody functions as the targeting module, we then replaced the IgG Fc domain into Coh2, a protein that interacts with DocS constitutively. Also, we truncated the Trim21 protein by removing the PRYSPRY containing 268-475 aa and termed the remining 1-268 aa of Trim21 the functional module. The functional module was then fused with a DocS protein on its C terminus to interact with the Coh2 fused GFP nanobody to achieve GFP targeting.

Test of replaceability of interface

With the above mention plasmids, wetlab experiments were performed to validate our design. With GFP as the target protein, we constructed the GFP PrePro plasmid: a plasmid expressing both Trim21-DocS and GFPnanobody-Coh2. Then, HEK-293T cells were co-transfected with GFP PrePro plasmid and GFP expressing plasmid. Fluorescent imaging and western blotting analysis showed significant decrease of both GFP fluorescent (Figure 2B and C) and GFP protein abundance (Figure 2 D). Also, we also observed that the GFP PrePro mediated GFP degradation lasts for at least 144 hours.These results suggested that replacing the PRYSPRY-IgG Fc interaction with DocS-Coh2 interaction would not impair the function of both targeting module and functional module, indicating that such new interface have been established for signal-control purposes.

 

 

Figure 2.GFP Predator Pro system based on the DocS-Coh2 interaction module. (A) Schematic representation showing the modularized design and function of GFP Predator Pro (GFP PrePro.) (B) Schematic representation showing the flow chart of dosage optimization and its results. The fluorescent intensity of GFP in each well was shown with heatmap. (C) Fluorescence images and intensity quantification of the GFP-Predator Pro transfected group and its negative control transfected with an empty vector. HEK293T cells in both groups were transfected with GFP-expression plasmid. (D) Western blotting determining the expression level of GFP in HEK293T cells transfected with GFP Predator Pro. (E) Model of degradation effect over time. (F) Fluorescence images showing the degradation of GFP in GFP Predator transfected groups at different times after transfection. N, P represents Negative control and GFP Predator, respectively.

 

Learn and Improve

With the experiment results as proof of concept, we presented our work on CCIC(click here for links to CCIC)and interviewed experts for advice.(click here for information about experts interview)From the feedback we learned that on the one hand, our approach seems promising, it seemed okay for us to move forward towards a signal-controlled system. But on the other hand, it was still necessary to normalize the influence that transcription efficiency other factors on GFP abundance. Therefore, we switched the reporting system from GFP to GFP coupled dual luciferase assay to achieve higher experimental stability. Also, further literature reading suggested us that we could try some well-established signal controlled protein dimerization pairs like rapamycin inducible FRB-FKBP pair in order to further prove our concept.

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