Team:NUDT CHINA/Engineering
Engineering
In our iGEM 2021 project, tremendous amount of engineering efforts has been made to bring the light responsive features into the system we kept working on for years. This year, by evaluating the degradation effects and interviewing some experts for improvement advice, we constructed a light-inducible protein degradation system that works in high efficiency. Here in this Engineering Success page, we will discuss the design and build, test and learn, and re-design process during the development of this system.
Figure 1. Engineering flow chart of constructing LiPrePro system.
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. In 2020, we have improved it as PREDATOR PRO system to achieve direct signal responsiveness by designing a proper interface to rewire the signal induced protein-protein interaction or other biochemical reactions. Moreover, in iGEM2021, we stepped forward upon a protein degradation system directly controlled by blue light.
According to the experience over the past few years of the mechanism of the interaction of Trim21 and targeted protein, we intended to replace the core module of PREDATOR PRO system into light sensitive Cry2/CIB1 pairs to develop a light inducible PREDATOR (LiPrePro) system. In this system, truncated Trim21 protein was linked with Cry2 with a GGGSG protein linker, targeting module was liked to CIB1 in the similar manner. 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 . In this way, we build a light inducible PREDATOR system.
To evaluate the light responsiveness of such system, HEK-293T cells were co-transfected with GFP expressing plasmid and LiPrePro plasmid/empty vector. Fluorescent imaging were captured 48 h post blue light illumination. Images showed a slight decrease (~5%) of GFP fluorescence in LiPrePro expressing group comparing to the control group kept under dark condition (Fig. 1B and 1C), indicating that our system couldn’t work as expected.
Figure 2. First trial 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).For (B-C), cells were illuminated with 3mW/cm2 of 405nm blue light, the illumination was programmed as the repeat of [2 s ON/58 s OFF] cycle for indicated hours. Statistical significance was calculated via student t test. *** p ＜ 0.001.
In the interview we had with Mirta Viviani, a phD student focusing on mammalian cell synthetic biology in Westlake University, we were informed that the potential problem could be related to the linker length between protein domains, which may dramatically affect protein folding.
Building on the advice from Mirta, we redesigned our plasmids, replacing the 1x GS linker into 3x or even 5x GS linkers(Fig. 2A). After cells were transfected with these new plasmids, much lower GFP level were gladly observed. Indicating 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, suggesting an improved protein degradation ability of these designs (Fig. 2B and C).
Figure 3. Optimization of light inducible PREDATOR system. (A) Schematic representation showing the design of 3x and 5x GS linkers. (B) Representative fluorescence images of the LiPrePro (3x GS) transfected group, LiPrePro(5x GS) transfected group and negative control group transfected with an empty vector. (C) Quantification of fluorescent intensity in (B). For (B-C), cells were illuminated with 3mW/cm2 of 405nm blue light, the illumination was programmed as the repeat of [2 s ON/58 s OFF] cycle for indicated hours. Statistical significance was calculated via student t test. *** p ＜ 0.001.
With such iteration process, we successfully developed a light inducible protein degradation system with decent degradation efficiency.