In order to choose a suitable project and ensure the scientificity, practicality and logic of the entire project, especially the rationality involved in medical ethics, we are involved in the selection, design, evaluation and final application stages of the entire project, We explored the opinions of professors in different professional directions. At the same time, we visited hospitals and obtained profiles of doctors. We sought their opinions, and their opinions were of great help in adjusting the entire project. At the same time, we designed a complete questionnaire based on the selected projects, and the feedback from the questionnaire provided opinions for the improvement of our projects.
As a first-year team, we weren't able to get a lot of help from the university. All the team members gathered together because they liked it. From the first time the initiator sent an invitation in the circle of friends, people from different professions gathered together. The winter in Northeast China is very cold, but everyone enthusiastically came to the seminar room to provide opinions on the construction of the entire team. This is a good platform that brings many students together. The team building has gone through several rounds of iterations, and many people have entered and left the team, finally forming the team we have now.
At the same time, Zayn Yang founded SYPHU's first iGEM club, which provided an opportunity for the team to hold activities. At this point, the construction of the entire SYPHU-China2023 team was completed. Of course, since it was the first year to participate, we did not do well enough in the HP part and did not have any particularly big innovations, but these activities still brought us a lot of feedback and suggestions to optimize our projects.
In this section, we will introduce an overview and breakdown of how our activities resulted in suggestions and how these suggestions optimized our project. These activities record our struggles, inspirations, and breakdowns throughout the project process. The final solution to these problems was achieved through the efforts of the entire SYPHU-China team.
This project was originally used for medical treatment, so we investigated many medical ethics issues. We also went to the hospital to interview doctors to understand the current situation and the issues that patients were most concerned about. Our PI and his friends helped us a lot, we communicated with them and improved our project, they helped us a lot.
Whether based on the value pursuit of our team or the high respect and attention to the results of the competition, we believe that a successful project needs to meet the interests and orientation of all stakeholders as much as possible. Therefore, our team fully referred to the suggestions of various stakeholders from the very beginning. In the project topic determination stage, after expert consultation, teacher guidance, literature review, public feedback, team communication and other processes, we comprehensively considered and excluded the infeasible ideas proposed in the "brainstorming". In addition, after combining the relevant principles of biological ethics, the project title was finally determined----oxygen hunter.
We paid attention to the particularity of the tumor microenvironment, with low oxygen and high lactate concentration. We hope to target this tumor microenvironment so that targeted delivery of drugs can be achieved. We noticed EcN, a strain that does not secrete any pathogenic E. coli enterotoxin and cytotoxin and is often used in the development of live biopharmaceuticals. Therefore we plan to use EcN as the chassis creature.So how to target this tumor microenvironment has become a key point. We were pleasantly surprised to find that EcN, as a facultative anaerobic bacterium, can target the hypoxic tumor microenvironment to a certain extent and, as an antigen, can induce the immune system to attack this site. We set our sights on targeting high lactate concentrations. Our focus is on the research on the Helicobacter pylori TlpC protein published by Mayra A. Machuca et al. in 2017, which confirmed that this is a protein that mediates lactate chemotaxis. Therefore, we focused our research on how to promote TlpC to mediate the lactate chemotaxis of EcN. By reviewing the literature, we learned that different strains have different chemotaxis pathways. The chemotactic pathways of EcN and Helicobacter pylori share some similarities. For example, they all use the group of proteins CheW, CheA, and CheY to mediate CheY phosphorylation through cascade translation to control the forward and reverse rotation of the flagellum. However, their methylation feedback proteins are different, with Helicobacter pylori using CheVs and E. coli using CheB and CheR. If TlpC is used directly on E. coli, some problems will inevitably occur. For example, E. coli cannot stop random rolling when approaching a point with high lactate concentration, resulting in TlpC being unable to mediate EcN lactate chemotaxis. We think about the relationship between the structure and function of proteins. We know that part of various chemosensory proteins needs to be responsible for binding to ligand molecules outside the cell to sense chemical signals, while the other part needs to participate in cascade reactions inside the cell. Now that we have the structure of the extracellular portion of the protein that binds lactate, why not try a recombinant protein? Then what protein to recombine with becomes a question. There must be some chemosensory proteins on the surface of EcN, but they just don't have the ability to sense lactic acid. After discussion, we all agreed that if we could find the protein on EcN that is closest to TlpC in structure and recombine it - just like building Lego, maybe we could make EcN, which is originally unable to sense lactate, chemoattract lactic acid. Through TlpC BLAST in the EcN genome, we found the Tsr protein with the highest similarity. By analyzing the structures of Tsr protein and TlpC protein using bioinformatics methods, we found that they are both twice-spanning membrane proteins, which is undoubtedly exciting news. So we replaced the extramembrane cytoplasmic binding domain part of Tsr, which itself binds serine, with the part of the extramembrane cytoplasmic binding domain of the TlpC protein. But we are also thinking that the intramembrane protein part of EcN must retain Tsr, and the extramembrane part must use TlpC. What about the transmembrane part? So we designed two proteins, eTlpC-A and eTlpC-B, one retaining the transmembrane part of TlpC and the other retaining the transmembrane part of Tsr. All were constructed for comparison, and finally the protein we wanted to use was determined.
After having strains with chemotaxis to lactic acid, we turned our attention to how to effectively kill tumor cells. Although EcN is an antigen, its accumulation in the tumor area will recruit the immune system to kill the tumor to a certain extent. But this effect is obviously not enough, especially since EcN is also a probiotic. We began to hope that EcN would directly secrete cytotoxins to kill tumor cells. But the idea was quickly dismissed because drugs without good specificity would cause strong side effects. We think of antibodies again, which can target some targets very well. We focus on the HER2 target. Trastuzumab, which targets HER2, is one of the earliest monoclonal antibody drugs to appear, and its mechanism has been thoroughly studied. So we choose it. We initially wanted bacteria to directly secrete antibodies to kill cancer cells, but this idea was obviously unrealistic. Because bacteria are incapable of secreting large molecules such as intact antibodies, this can lead to the inability of the antibodies to fold correctly. At this time, we noticed the single-chain antibody technology. Due to its relatively small molecular weight, this antibody can be expressed in a prokaryotic expression system. We retrieve its structure in NCBI, preserving its VH and VL parts. After that we use linker to connect it. At this time we encounter a problem, is VH first or VL first? What linker to use? By reviewing the literature, we learned that different sequences have different tendencies. VH in the pre-antibody has a strong affinity, and VL in the pre-antibody has a high secretion amount. We found that there are marketed drugs for both connection methods. We chose the VL first connection method to ensure secretion volume. Use GGGGS, a linker widely used in single-chain antibody construction, to connect them. However, single-chain antibodies have lost the Fc segment, so they have no ADCC and CDC effects. So how to complete the important task of killing tumor cells? We thought of missiles. Why not use this single-chain antibody as a missile head to guide some non-specific cytotoxic agents to kill tumor cells? We couldn't be more excited. But there is a question, which cytotoxic agent to choose? We noticed that the molecular weight of single-chain antibodies is too small, and most people stabilize it by increasing its molecular weight by coupling it with serum albumin, for example. Ordinary ADC drugs are small molecules coupled to intact antibodies. Why don't we combine it. We think of protein toxins. Through searching in the drug bank, we found that there is a similar drug, Moxetumomab pasudotox, which is already on the market, which further supports our conjecture. If a protein poison can be used, it can not only stabilize the single-chain antibody, but also give the single-chain antibody the ability to kill target cells. We selected PE38 as the conjugated protein cytotoxic agent with reference to Moxetumomab pasudotox. At the same time, we found that HER2 is a target with good internalization, which undoubtedly enhances our confidence. Making ADC drugs undoubtedly requires linkers. In front of us are flexible linkers like GSSSS and rigid linkers like EAAAK. Since the function of a linker is to isolate different domains of a protein so that they can function independently, we tend to use rigid linkers like EAAAK to isolate antibodies and protein toxins. But we also tried GGGGS. With ADC drugs, how do we express it. Constitutive expression? This idea was quickly rejected by us. Even ADC drugs are not 100% specific. Moreover, constitutive expression will bring unnecessary risks in the experiment and cause harm to the growth of the strain. We plan to use some inducible promoters, so what factors should be chosen for induction? We also thought about the tumor microenvironment, which is high lactic acid and low oxygen. We chose a lactate-inducible promoter because this is also our chemotactic factor. We found PlldR, a lactate-regulated promoter. However, we are worried that the expression intensity of wild-type PlldR is not strong enough to express a sufficient concentration of ADC drugs. We changed its promoter region to a T7 promoter and introduced the T7 RNA polymerase gene into EcN to enhance protein expression. However, we found that due to the strong expression activity of the T7 promoter, its leakage expression was abnormally high, which was undesirable. We also found this phenomenon in other studies. Strong promoters are often accompanied by strong leaky expression. So we thought, since we can't control the T7 promoter, how about we control the T7 RNA polymerase that controls the T7 promoter. By simultaneously controlling the expression of T7 RNA polymerase and the T7 promoter, is it possible to achieve high protein expression while maintaining low leakage expression? We achieve this by using wild-type PlldR to control T7 RNA polymerase, and using T7-modified PlldR-T7 to control ADC drugs.
We compiled the above functions into a gene circuit. We once considered using plasmids as expression vectors to locate the gene circuit on the plasmid. But then we considered that the presence of the plasmid was bound to be accompanied by a selection tag, which would lead to some antibiotic resistance in our strains. this is not good. Because we want to use the strain for human use, we do not want it to carry plasmids with resistance genes, which will make it difficult to kill and cause risks. It can also easily cause horizontal gene transfer, causing greater risks, not only to humans, but also to environment. Therefore, we decided to knock the genetic circuit into the EcN genome so that we could get rid of the resistance gene. We also plan to add a suicide gene to it to ensure the safety of the drug. At this time we encountered a problem, what inducing factors to choose. We focused on the Cas system we used, which has a pBAD promoter and its inducer is arabinose. At the same time, we noticed that the iGEM distribution plate contains an optimally regulated gene for this promoter. We thought, this is it. We discovered that arabinose is also a health supplement, so we were even more determined to use it as an inducer for our suicide switch. When selecting suicide genes, we hope that this protein will only kill our strain and not affect other cells in the body. Therefore, we chose the protein ccdB, which only inhibits bacterial DNA gyrase and causes apoptosis, but has no effect on eukaryotic cells. So Our project produced!!!!!!
The overall style of the team uniform design is simple and elegant, with navy blue as the main base and the blue team emblem on the left chest, which symbolizes calmness and rationality, and also shows that the team upholds the school's spiritual connotation of "perseverance and medicine for the world"; The two sleeves are white with a dark simple DNA double helix model printed on them, which expresses its biological theme; at the same time, the words "innovate" on the right side of the team uniform on a white background refer to the school motto of "Unity, Diligence, Pragmatism, and Innovation". It explains the core element of the team - innovation; the double helix structure and cell pattern on the back, with philosophical text below, further highlight the content theme. Overall, I hope that the design of this team uniform can further bring the relationship between igem team members closer, improve team cohesion, give everyone a deeper understanding of the theme of scientific research and innovation, and strengthen cooperation.
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The repository used to create this website is available at gitlab.igem.org/2023/syphu-china.
