Team:NUDT CHINA/Results
Results
Constructing Blue Light Illumination System & Selecting Protein Dimerization Pairs
The first step in constructing the Cycle Blue system is to build a blue light illumination system which can manually adjust the light intensity and illumination time. The device was built with 4 sets of blue light LEDs (6 LEDs each, 405nm, 1-3W) glued on an aluminum heatsink. The illumination device was covered by an acrylic light shield, with non-transparent surroundings and a clear top. Four standard cell culture plates could be placed on the top of the illumination device. Each LED sets were controlled independently with electromagnetic relays under control of a Raspberry Pi single-board computer (Fig.1A). The Raspberry Pi was loaded with ubuntu system and a python-based control script, allowing us to control the on/off time of each set of LEDs conveniently (Click here to see our Hardware page and know more about the blue light illumination system design)(Fig.1B)
Figure 1.Construction of blue light illumination system. (A) Schematic representation of the system design. (B) Pictures of the blue light illumination system installed in the incubator.
During our discussion with Professor Yang regarding the light-inducible protein pairs, we were suggested to use online databases (like https://www.optobase.org/) to identify potential optoswitches that could function as core module (interface: in IGEM 2020 NUDT_CHINA) to generate a set of light controlled protein degradation tools. Herein we chose Cry2/CIB1, a protein pair that dimerize under blue light, and other two protein pairs that dissociate under blue light: LOV2/zdk1 and PixD/PixE. To verify the fitness of these interaction pairs to our illumination device and our chassis, we inserted the candidate protein pairs into a tetR-based system. Whiles one member of the protein pair was linked to a tetR DNA binding domain, the other member of the protein pair was linked to a nuclear localized transcriptional activation domain VP64. Upon co-transfection of these plasmids with a tetO7 promoter driven SEAP reporter, we would be able to evaluate the binding affinity and light responsiveness of the interaction pairs by measuring SEAP activity in the culture medium (Fig.2A).
As expected, HEK-293T cells co-transfected with tetR-Cry2, CIB1-VP64 and tetO7-SEAP expressing plasmids showed significantly increased SEAP production upon 24h or 48 h of blue light illumination comparing to control cells transfected with the same plasmids placed in a dark incubator (p＜0.001, Fig.2B). Also, in consistent to previous reports, cells co-transfected with tetR-Lov2, Zdk-VP64 and tetO7-SEAP plasmids showed significant reduction of SEAP production upon either 24 h or 48 h of blue light illumination comparing to the control cells (p＜0.001, Fig.2C). To our surprise, low SEAP production was detected in cells co-transfected with tetR-PixD, CIB1- PixE and tetO7-SEAP expressing plasmids under either light or dark conditions (p＜0.001, Fig.2D). Considering the absolute level of SEAP production was way higher in Cry2/CIB1 pair comparing to the lov2/zdk pair, these results indicated that Cry2/CIB1 suits our illumination hardware and parameter the best. Therefore, Cry2/CIB1 pair was then used in our next phase of experiments.
Figure 2. Validation of the blue light-induced pairs. (A) Schematic representation showing the function of light inducible protein pairs and design of Pairs-TetON system. (B) SEAP activity in the culture medium collect from cells transfected with tetR-Cry2, CIB1-VP64 and tetO7-SEAP expressing plasmids under either blue light illumination or dark condition. (C) SEAP activity in the culture medium collect from cells transfected with tetR-lov2, zdk-VP64 and tetO7-SEAP expressing plasmids under either blue light illumination or dark condition. (D) SEAP activity in the culture medium collect from cells transfected with tetR-PixD, PixE-VP64 and tetO7-SEAP expressing plasmids under either blue light illumination or dark condition. For (B-D), cells were illuminated with 3 mW/cm2 of 405nm blue light, the illumination was programed as the repeat of [2 s ON /58 s OFF] cycle for 48 hours. BDL represents below detection limits, Data represent mean ± s.e.m. (n=3 biological replicates); ***P ＜ 0.001; two-tailed unpaired Student’s t-test.
Construction and Optimization of Blue Light Inducible PREDATOR System
To develop a light inducible PREDATOR (LiPrePro) system, we intended to replace the core module of PREDATOR PRO system into Cry2/CIB1 pairs. In order to simplify the test and optimization procedure, GFP was used as the target protein, therefore a simple fluorescent imaging could be sufficient to evaluate the degradation efficiency (Fig.3 A). To begin with, truncated Trim21 protein was linked with Cry2 with a GGGSG protein linker, GFPnanobody was liked to CIB1 in the similar manner. As a validation of such system, HEK-293T cells were co-transfected with GFP expressing plasmid and LiPrePro plasmid/empty vector. Fluorescent imaging showed slight decrease (~5%) of GFP fluorescence in LiPrePro expressing group comparing to the control group (Fig. 3B and 3C) under 48 h blue light illumination, which was unsatisfying.
During our discussion with Mirta Viviani, a phD student working on synthetic biology in Westlake University, we mentioned the preliminary failure in generating satisfying protein degradation under blue light control. She pointed out that we could try optimizing our system structure, such as adjusting the linker length between modules to help proteins folding correctly. Therefore, we changed the length of linker between Cry2 and GFPnano and the linker between CIB1 and truncated Trim21 from 1x GS linker into 3x or even 5x GS linkers (Fig.3D) (Click here to see our design page for more information). Results showed significant reduction of GFP levels in cells transfected with either LiPrePro (3x GS Linker) or LiPrePro (5x GS linker) comparing to the EGFP transfected control cells under 24, 48 and 72 h of blue light illumination, indicating an improved protein degradation ability of these designs. (Fig. 3E and F).
Figure 3. Construction and Optimization of light inducible PREDATOR system. (A) Schematic representation showing the design of Blue light-inducible Predator Pro (LiPrePro) system. (B) Representative fluorescence images of the LiPrePro transfected group and negative control group transfected with an empty vector. (C) Quantification of fluorescent intensity in (B). (D) Schematic representation showing the design of LiPrePro with 3x and 5x GS linkers. (E) Representative fluorescence images of the LiPrePro (3x GS) transfected group, LiPrePro(5x GS) transfected group and negative control group transfected with an empty vector. (F) Quantification of fluorescent intensity in (E). For (B-C, E-F), cells were illuminated with 3 mW/cm2 of 405nm blue light, the illumination was programed as the repeat of [2 s ON /58 s OFF] cycle for indicated hours. Data represent mean ± s.e.m. (n=3 biological replicates); ***P ＜ 0.001; two-tailed unpaired Student’s t-test.
Targeting Cyclin and Control Cell Proliferation
As intracellular antibodies and ScFvs are the most obvious approaches for specific recognition of target protein, we first focused on finding the published ScFvs targeting specific cyclin protein. To simplify the experiments, we kept the original DocS/Coh2 core module of the PREDATOR system. With this constitutively binding core module, we expect to see significant reduction of cell proliferation in Cyclin PREDATOR transfected cells with either microscopic imaging or CCK8 assay (Fig.4A).
With the Cyclin E specific ScFv sequence published by Y Wu, we designed multiple ScFv or split ScFv based Cyclin E PREDATORs(Click here to see our design page for more information). To validate these designs, HEK-293T cells were transfected with PREDATOR expressing plasmids, CCK8 viability assay was performed 36 h post transfection to evaluate the cell proliferation. While results showed significant reduced cell proliferation in most of these designs (Fig. 4B and C) the reduction of cell proliferation was unsatisfying.
In the meantime, two other targeting modules were designed under the suggestions from Prof. Chun Meng(Click here to see our design page for more information). Herein, we obtained the αCHelix region of CDK4 and Rb, which were the endogenous binding proteins of Cyclin D1. Using the tandem repeats of these Cyclin D1 binding regions, we designed new Cyclin D1 PREDATORs. Results showed significantly reduced proliferation in cells transfected with either designs, indicating tandem repeats of CDK4 αCHelix or Rb αCHelix could be considered as an optimal targeting module.
Figure 4. Optimalization of Cyclin-targeting module. (A) Schematic representation of the experimental process of cyclin PREDATOR validation. (B,D) Representative microscopic images of HEK-293T cells transfected with indicated plasmids. (C,E) CCK8 cell proliferation assay of HEK-293T cells transfected with indicated plasmids. For (C and E), data represent mean ± s.e.m. (n=3 biological replicates); ***P ＜ 0.001; two-tailed unpaired Student’s t-test.
Light-mediated control of mammalian cell cycle
With the optimal protein-protein pairs, optimization of linkers and Cyclin-targeting module, we then build a blue light inducible Predator system to mediate the regulation and intervention of the cell cycle as a proof of our concept. Integrating with a raspberry pie-based blue light illumination device, light-controlled dimerization of protein pairs may occur and subsequently ternary degradation complex formed, leading to the degradation of Cyclin (Fig.5A). Herein, we designed two CycleBlue systems namely CycleBlue-1 and CycleBlue-2, using CDK4 αCHelix or Rb αCHelix as targeting module, respectively.
For characterization, HEK-293T cells were transfected with either CycleBlue-1 or CycleBlue-2 expressing plasmids and illuminated with blue light 24h post transfection. Cyclin levels and cell proliferation were analyzed by Western Blotting and CCK8 assay, respectively. Intriguingly, under blue light illumination, western blotting showed significantly reduced cyclin D1 level in cells transfected with CycleBlue-1 comparing to the control group, while the reduction on cyclin D1 level in cells transfected with CycleBlue-2 was not observed (Fig. 5B). Interestingly, CCK8 assay(Fig. 5C) showed significant reduction of cell proliferation under blue light illumination in both CycleBlue-1 and CycleBlue-2 transfected cells. (Fig.5C, right panel, p ＜ 0.001), while the effect of CycleBlue-1 and CycleBlue-2 transfection in dark condition was insignificant (Fig.5C, left panel, p ＜ 0.001).
In general, these results showed that our CycleBlue system can successfully degrade Cyclin D1 and regulate cell proliferation under the control of blue light with decent performance. The engineering success of these designs also showed the potential of CycleBlue system to be further developed into a novel synthetic biology toolbox for controlled protein degradation.
Figure 5. CycleBlue: Blue-light controllable cell growth. (A) Schematic representation showing the design of CycleBlue system. (B) Representative Western blotting determining the Cyclin abundance in HEK-293T cells 48 h post transfected with CycleBlue-1 plasmid and CycleBlue-2 plasmid under blue light illumination condition. (C) CCK8 cell proliferation assay on CycleBlue-1/ CycleBlue-2 transfected HEK-293T cells under dark or blue light illuminated conditions. For (B-C, E-F), cells were illuminated with 3 mW/cm2 of 405nm blue light, the illumination was programed as the repeat of [2 s ON /58 s OFF] cycle for indicated hours. Data represent mean ± s.e.m. (n=3 biological replicates); ***P ＜ 0.001; two-tailed unpaired Student’s t-test.