Contribution

Overview

This year, in order to realize the fundamental concept of NanoDisguiser, BNU-China has made efforts in multiple areas. We enriched the iGEM parts library, conducted extensive exploration in protein purification, and investigated sample preparation methods for nanodiscs, a special material, to observe them under transmission electron microscopy. The different protein linkage methods we used can also serve as a reference for other research.

BNU-China is committed to building a more rational and inclusive society, with its own plans for enterprise development.

In the process of advancing our project, we utilized comprehensive and complete computer-aided simulations. We sincerely hope that these efforts can inspire and assist future synthetic biologists and iGEM teams. We aim to promote the idea of nanodisc antiviral applications, contributing to the prevention or treatment of infectious diseases and raising awareness about the infinite potential of nanodiscs.

Part1 Enriches the iGEM parts library

Based on the highly versatile and promising nanodisc tools, we have developed multiple MSP parts for constructing nanodiscs of various sizes, including MSP1E3D1, spMSP1D1, spNW50, and spNW15. Based on these, we used the spy/sdy/snoop linkers and further crafted flexible and length-tunable multi-polymerized MSP. After further optimization, we designed the production of self-cyclized MSPs and multimeric cyclic MSPs of varying lengths in the same engineered bacteria. Additionally, the introduction of split-mCherry offers fresh inspiration for characterizing protein interactions.This provides scientific researchers and future teams with intriguing building blocks for creating large-scale nanodiscs, and also introduces a "logic gate"-like concept for protein cyclization or interaction.

We propose the idea of receptor conjugation using the split-GFP two-component technique. We use the biotin-streptavidin system to tether the biotinylated membrane protein to sGFP, then leverage the interaction between sGFP1-10 and sGFP11 to link two membrane proteins. In the area of protein linkage, these methods may provide inspiration for other teams.

Regarding functional validation, we have modified the SEND system developed by the Zhang Feng team to enable fluorescence-based representation of drug defense capabilities, presenting a safe and intuitive new approach for future pre-clinical drug testing. This module is now offered as a new part for iGEMers to utilize.

In summary, our team has contributed a diverse and innovative set of new parts to the iGEM community, as well as introduced promising nanodisc tools with broad applications in medicine, membrane protein research, and beyond. We hope this work serves as a valuable reference and offers enriching options for future teams.

For more details, please refer to our Parts page.

Figure 1: MSP structure prediction

Part2 Optimize the expression conditions of special proteins

The process of producing nanodiscs involves purifying a range of proteins, including four monomeric MSP proteins, three polymerized MSP proteins, and two split-GFP adaptor proteins. While the production and purification methods for some of these proteins are well-documented in the literature, the innovative proteins we developed lack corresponding literature references. We investigated a series of experimental conditions, optimized the purification methods for the former group of proteins, and established purification systems for the latter.

Key factors in protein purification include the concentration of the inducer IPTG, induction temperature, the choice of protease inhibitors and so on. We meticulously examined each condition. For instance, E.coli was induced with IPTG at various concentrations, and different protease inhibitors were employed for specific proteins. Ultimately, we identified the optimal purification conditions for each protein.

It is important to note that MSP proteins are amphiphilic, existing between soluble and insoluble proteins. As a result, the purification conditions for MSP proteins are more specialized than those for typical proteins. Our findings will provide valuable insights and frameworks for future teams aiming to purify MSP or similar proteins.

Part3 Exploration of sample preparation for negative stain electron microscopy

The characterization of nanodiscs in our project involves the use of negative stain electron microscopy. Negative stain electron microscopy is an electron microscopy technique used to observe biological samples, particularly suitable for observing small biomolecules that are difficult to study through traditional ultra-thin sectioning techniques, such as proteins, nucleic acids, viruses, etc. This method uses heavy metal salts (such as phosphotungstic acid, uranium acetate, etc.) as dyes to stain the sample, creating contrast under an electron microscope and allowing the morphology and structure of the sample to be clearly displayed.

It is crucial to observe sample preparation in negative stain electron microscopy. We used a 300-mesh copper mesh with a carbon support film, and compared to uranyl acetate with a small amount of radioactivity, we chose phosphotungstic acid to stain the sample. After multiple attempts, we have developed a detailed and feasible method and found that the dispersion of the sample and the integrity of the copper mesh are particularly important for the electron microscopy observation effect (For specific content, please refer to our Notebook page). For special materials such as nanodiscs, we have also explored a feasible and more convenient sample preparation method. We hope that our attempts and experience can provide assistance to the igem team that needs to use negative stain electron microscopy in the future.

Part4 Computer-assisted simulation and experimental design assistance guide the experiment

In our project, computer simulations were used several times to assist and guide our experiments.

We used AlphaFold to predict the MSP protein structure and the protein structure after directed evolution, which helped the experimental group to exclude unreasonable designs. We creatively used the infectious disease model to verify the function of nanodiscs and improve the experiments. For experiments in the future, we used computer-guided directed evolution and molecular docking simulations of evolved membrane proteins, hoping to further optimize the function of nanodiscs.

We use pharmacokinetic simulations to predict the ultimate diffusion and metabolism of nanodiscs in the human body.

Due to the limitations of the actual situation, our experiments are often faced with many difficulties. Modeling used the experimental design method to guide experiments.The experimental design can take into account all the factors, and give the best design in different situations, so that the data obtained from the experiment are of high quality and conducive to subsequent analysis. In our project, considering the limitations of the number of experiments and economic reasons, we gave the orthogonal table with nested design, which solved the difficulties in the experiment in time and achieved good results.

Part5 Building a more rational and inclusive society

This year,we have focused on the tangible benefits that synthetic biology can bring to each individual and convey this concept to the public, hoping that more people can know, understand, and eventually identify with it.Our project has raised public awareness of infectious disease issues and enhanced people's self-protection and prevention awareness. In addition, we have promoted public understanding of special populations (specifically, visually impaired, hearing-impaired, and autistic) and achieved significant results in eliminating biases and misunderstandings. Finally, we have striven to overcome obstacles and prevent distance or language from becoming barriers to scientific communications. We have identified specific needs of different audiences, tailored knowledge dissemination methods for them, and received fantastic feedback. More details can be found in our Inclusivity page.

We have established long-term cooperative relationships with institutions such as Braille Library, Xiehe Fangtong Autism Rehabilitation Center, Shuyun Hearing Rehabilitation Center, as well as multiple teaching teams from Beijing Normal University. We will continue to monitor the situation of special populations and children in remote areas who have been encouraged by us during the 2024 iGEM period, constantly discovering their needs, so that future iGEM teams can be more targeted in their work without starting from scratch.

We have produced comic booklets, popular science videos, and fun mini games that are aimed at all kinds of populations and permanently effective. They will continue to be disseminated online or offline, benefiting more people. We strongly welcome future iGEM teams to reuse and recreate our products, and together we will spread science and love to farther places.

Part6 Referenceable train of thought for entrepreneurship

For this year's truly new project, we have planned the transformation from 'laboratory' to 'company' at the very beginning. And in this process, we have accumulated a lot of experience and lessons. We have learned to analyze existing business models, improve and enhance them, so that they can be closely integrated with the needs of our project itself. Due to the rarity of teams preparing for entrepreneurship at the beginning of project design, we consider it a pioneering work. In addition to exploring on our own, we have also consulted experts from various fields for advice and recorded our thoughts and growth throughout the entire process in detail. If the subsequent iGEM teams also want to industrialize the project but lack relevant experience, they can learn from our work and innovate accordingly. We believe that our explorations will definitely bring them infinite inspirations!More details can be found in our Entrepreneurship page Entrepreneurship page.