As mentioned in the project introduction, our team has made a lot of efforts to achieve the project design and we hope to provide broader perspectives for future iGEM participants and create more possibilities for future iGEM projects. We have introduced brand new components and conducted scientific measurements and evaluations on them. We have also provided new operational methods and experimental techniques for future iGEM projects, which will simplify the experimental work for future iGEM teams. Additionally, we focus on education and pay attention to different social groups, aiming to promote the core principles of synthetic biology and expand its reach to a larger world.
In this year’s project, we have designed multiple brand new proteins. We needed to constantly experiment with different optimal conditions for protein expression and purification. However, through our painstaking efforts, we ultimately succeeded in expressing and purifying four different proteins and measured some of their properties, which will provide valuable references for future iGEM teams using our parts. For the specific composition and functionality of each component, please refer to our part wiki.
We have developed a purifying method that is more suitable for our proteins. It simplifies the protein extraction process, improves purification efficiency, and successfully solves the problem of removing endogenous biotin.
The prototype of our hardware model is a nickel ion chelation column. At the beginning of the project, we added the His tag to the Silinker protein for purification and used nickel affinity chromatography. However, to obtain each Silinker protein, we needed to enzymatically cleave the tagged protein to expose the mSA site.
Figure1| Remove endogenous biotin
Extracting the Silinker protein from the mixture after enzymatic cleavage was a challenging task. Additionally, we wanted to expose the mSA site and remove endogenous biotin that had already bound to the Silinker protein. To achieve this, we needed to immobilize the Silinker protein for biotin elution, which required heating the protein at 75°C. Unfortunately, Ni-NTA His-Tag purification resin is unable to withstand such high temperatures. This means that using traditional methods would not efficiently purify our Silinker protein. Therefore, we have created a new, cost-effective, convenient, and reusable silica protein purification hardware utilizing the affinity between the SBP sequence and silica. It simplifies and enhances the purification process of our Silinker protein, and we have already validated its feasibility.
After exploring multiple conditions, we made the final discovery that effective elution of Silinker protein, which is already bound to silica dioxide, can be achieved under the following conditions: 2M lysine, 700mM NaCl, pH=9.0, and 0.3% Tween in the Elution Buffer.
Principle:
Silica-binding peptide (SBP) has a high affinity for binding to silica dioxide. This interaction can be disrupted by L-arginine or L-lysine. After conducting a literature review, we attempted to use L-lysine for elution, but unfortunately, it was not successful. Therefore, we optimized the elution solution through multiple trials and finally developed an elution solution suitable for our protein. Effective elution was achieved by reducing the concentration of lysine.
We performed optimized small-scale purification under high salt and high pH conditions and obtained purified protein. The combination of the aforementioned features allows for inexpensive recovery of Silinker protein within minutes.
This technique is valuable for laboratory-scale applications requiring rapid acquisition of milligram-scale Silinker proteins and preparation-grade purification. Compared to traditional methods, it offers advantages such as lower cost and shorter processing time.
Figure 2| Work flow of the Silinker protein purification device
The Silinker protein purification device has significant application significance in this project. It consists of a plastic casing, SiO2 packing material, and a collection chamber. The Silinker protein purification column is equipped with 0.1mm pore size SiO2 packing material, which is used for conventional affinity purification of fusion proteins containing SBP short peptides.
Versatile
Suitable for proteins with SBP domains, not limited to Silinker family proteins.
Compatibility
Can be used with various buffer additives, suitable for both heat and denaturation applications.
Cost-effective
The silica packing material can be reused multiple times, resulting in low raw material costs.
Our hardware can be used for the purification of proteins carrying SBP domains, not limited to members of the Silinker family. Due to the high stability of Basic Silinker, proteins can be recovered and reused. The hardware can be used in conjunction with our reagent kit to remove functional proteins that have been attached to Basic Silinker through Monomeric streptavidin and biotin interactions. Additionally, the affinity between SBP and the silica surface enables the use of the hardware for enzyme immobilization, protein screening, and other experiments.
1.We have organized the first Conference of Beijing iGEMer Community (CBiC), which has been recognized by the committee. This will provide a platform for future Beijing iGEM teams to learn and make progress together, which will be a great opportunity to integrate iGEM teams in Beijing and even more regions. You can read more about it on our (Conference of Beijing iGEMer Community) page.
2.As Synthetic Biology is not a field that is typically encountered in primary, middle, or high school, we have prepared Synthetic Biology courses to introduce this field to more students and engage them in it. The content of these courses is simple but very attractive and has been carefully crafted. We believe that this will be a good educational resource. We provide all our course syllabuses and materials as a contribution to future iGEM teams. Click (here) to learn more.
3.To integrate knowledge of synthetic biology into popular life and avoid learning barriers caused by jargons and stimulate public interest in understanding synthetic biology, we have designed a Synthetic Biology game called BioFrame Riddles. We believe this game can serve as a reference for future iGEM teams to spread knowledge of synthetic biology. You can read more about it on the (BioFrame Riddles) page.
4.We have listed detailed steps and suggestions for how to prepare materials for education and inclusivity, such as (course syllabuses and materials), (podcasts), and (science videos) on the corresponding web pages for future iGEM teams to refer to and carry out their own science popularization activities.
5.We have designed 16 educational personality types and 4 dimensions of evaluating learning styles that allow us to determine how we should educate our target audience to obtain the best educational results. We have also included relevant questionnaires to help iGEMers determine more easily and more accurately. You can obtain more detailed information and our questionnaire on the (Education Personality) page.
6.We sought expertise from experts in special education, including those with hearing and visual impairments, and personally communicated with these disabled people to understand their learning difficulties and summarize educational guidelines for deaf children and visually impaired children. We also share the process of our educational activities for future iGEM teams to reference and implement, allowing them to optimize their education and inclucivity activities to implement their ideas more effectively. You can view related content on the (Deaf) and (Visual Impairment) pages.
7.Based on the sixth point, we have also created picture books for visually impaired children, which are touchable and scentable special Braille picture books that allow visually impaired children to experience biology without visual barriers, through touch or smell. You can view the full content of our picture book on the (picture books for visually impaired children) page and download the picture book illustrations. We have also written book-making tips for future teams to refer to and use.
After numerous experimental attempts and optimizations, we have created usage instructions for our protein, compiled into the Basic Silinker Binding Kit. We have also included detailed information about our protein. We have made every effort to ensure that the instructions in the reagent kit are clear and comprehensive, including the required materials, preparation steps, and relevant precautions. This will greatly reduce the work required for future iGEM teams when using our reagent kit and our part. We genuinely hope that this will be helpful and supportive to future iGEM teams.(Experiment)
We recognize the importance of protocols in the experimental process as they provide detailed and clear instructions for conducting an experiment. Therefore, we have compiled our experiment protocols for this year, making it convenient for future iGEM teams to use and replicate our experiments.
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