Assistance during experiment with LZU-HS-Pro-A
At the beginning of the experiment, team LZU-HS-Pro-A helped us prepare consumables, such as ddH2O, 0.1M CaCl2, and a liquid culture medium. Since our experiments are similar in several parts, we can save time by reducing repetitive procedures. Similarly, they had our plasmid extraction done, so we did not have to spend extra time there. During the experiment, the problem they faced is the lack of materials. They had an inefficient AMP to make AMP antibiotic mother solution, and they were stuck due to this limitation. At that time, our team gave them the excess AMP that we own to help them continue. We try our best to solve all the issues we met and try to experiment as effectively as possible.
Assistance during experiment with LZU-HS-China-A
We conducted the experiment in the same laboratory with LZU-HS-China-A as well. Firstly, we reminded each other about safety during the experiment. When using the clean bench, we needed to spray alcohol on the item that has to enter. Plus, when conducting electrophoresis, we must wear second gloves, to protect us from harmful chemicals. Then, when balancing the centrifuge, we checked each other’s balance again for safety concerns. Lastly, we shared the conference room, and we communicated the experience that helped us more familiar with the experimental apparatus, and also raised our awareness of safety.
Articles revise and improvement
Since English is a second language for us, we need to edit and correct our articles carefully to reduce the number of grammatical mistakes. Therefore, our team collaborated with team LZU-HS-China-A, to double-check and fix the language problems. We swapped our articles, and after reading meticulously, we pointed out all the mistakes we made, and tried our best to amend them. After this collaboration, we believe the quality of our content has been hugely improved.
Research and selection based on anchor protein used in cell surface display
Firstly, and most importantly, since we both use cell surface display technology in our projects, we can help each other technically. In the earlier phase, the problem we faced was choosing a proper anchor protein, which is an essential protein used in cell surface display technology. This is a critical section of the projects for our three teams, but we have no idea which kind of protein can be used, so during our first online meeting, we set up a detailed plan for our projects’ future progress.
Evidence of online meeting
We initially did some research based on the properties required for anchor proteins. By exchanging useful papers, then giving presentations to obtain a clearer understanding of anchor protein, we get the following information:
-Properties required for anchor proteins
1. Having signal peptide sequences or transport signals that guide the fusion protein across the cell membrane
2. Can guide the fusion protein to specific sites on the cell surface
3. Having stable anchor elements that can be recognized at specific sites on the cell surface, so that the fusion protein can be immobilized on the cell surface
4. Fusion with foreign proteins will not change its stability, and it is uneasily to be broken down by proteases in or outside the cell
In addition, we found out the consequences of choosing an unsuitable anchor protein:
1. The structure of anchor protein and passenger protein is easy to affect each other, interferes with their correct folding, and reduces the activity of passenger protein
2. Different transporter proteins will affect the physiological function of the recipient bacteria (growth instability, etc.)
After filtering these required properties, we obtained several satisfied anchor proteins:
Lipoprotein, S-layer protein and INP.
From them, also followed the instructions given by our advisors, we eventually decided to use ice-nucleation protein, since its advantages in cell surface display technology:
-Pros of INP
1. Stably expressed in a large variety of species of host cells
2. Uneasily to be broken down by proteases in or outside the cell
3. High efficiency of displaying on the surface of the host cell
4. Repeating structures can be cut without influencing the functions, which can be advantageous when carrying passenger proteins with larger molecular weights
5. No auxiliary proteins are required
-Cell surface display mechanism
Ice-nucleation protein is frequently used in cell surface anchors for protein display in Gram-Negative bacteria. The bacterial surface display has been widely applied in scientific research and the science industry. Various functional peptides, recombinant vaccines, and catalytic enzymes could be displayed on bacterial surfaces through appropriate surface anchors, which facilitated their further engineering and applications for certain purposes.
The hydrophobicity of the nitrogen terminal is fused with the phospholipid bimolecular membrane to fix the linked enzyme on the cell membrane, and the hydrophilicity of the carbon terminal is exposed outside the cell membrane to connect with the acting enzyme so that the enzyme can be effectively expressed outside the cell membrane.
Evidence of discussion and presentation
In the end, by experiments, the efficacious result proves that our discussion and collaboration are successful and vital. During this experience, collaboration allowed us to work more effectively by urging and encouraging each other. Also, we were able to interact with our ideas and brainstorm together, which is particularly beneficial for our projects’ promotion.
Projects popularization
During the middle stage of iGEM, another aspect of collaborating is publicizing our projects. In order to obtain more valid data for integrated human practice, we had to popularize our questionnaire and project introduction. However, due to the fact that these specialized topics are not that straightforward for people to understand, our WeChat official accounts have limited figures for pageviews, which is not conducive for us to appeal to more people, and raise their awareness of environmental issues (etc.) involved in our projects.
Thus, after holding an online meeting, we decided to let more people know us by promoting each other’s projects. We achieved this goal in two methods. Firstly, we summarized others’ project descriptions, make them less complicated, and published them in our account. During the discussion, we thought simplifying is a useful way to make our articles more readable. Plus, although we only have a small number of followers, the sum of followers of our three teams is still relatively sizable.
We can see remarkable progress in pageviews after collaboration, and we firmly believe that by our influence, more people are willing to consider these crucial issues. This significant increase also reveals our efficient collaboration, when facing a problem, that we can achieve our targets using a simple but useful method.
Information collection and preparation for synthetic biology education
After some time, when preparing for the section on education, we cooperated again for better performance on what we can contribute to the public and synthetic biology. We firstly divided the workload via online meetings, in which our team was in charge of presenting, LZU-HS-China-A for slides and poster creation, also online lecture, and LZU-HS-China-C for related content preparation. This work division was based on our advantages of owning a proper venue for education, outstanding design skills from LZU-HS-China-A, and LZU-HS-China-C who are good at information collecting.
Evidence of giving out posters and presenting
We accomplished this section together, not only for better effectiveness but also to allow our three teams to bring out our diverse strengths, to make a greater performance. Moreover, more audiences are involved because we separately propagandize the lectures. These advantages can be only shown between team collaboration, therefore we are able to achieve a better job.
References
1. Li Qixi. (2009) Construction of bacterial cell surface display system using ice crystal nuclear protein and its application. Huazhong Agricultural University.
2. Zhang, Z. et al. (2016) Surface immobilization of human arginase-1 with an engineered ice nucleation protein display system in E. coli, PloS one. Public Library of Science.
