In the process of reading the literature, we learned the shortcomings of physical, chemical and enzymatic methods. When chitosan oligosaccharide is hydrolyzed, it is difficult to determine the polymerization degree of oligosaccharide and the pollution of the environment by using physical and chemical methods; Enzymatic method can determine the degree of polymerization, but it will lead to low efficiency, complex separation and purification, and immobilization is easy to inactivate; The cell surface display technology we finally selected provides the possibility to display larger molecules, including active enzymes. In addition, adsorbed peptides or proteins can also be displayed. Common display systems include outer membrane protein C, ice nucleation protein and INP variant.
Enzymes displayed on the surface of bacterial cells can make the enzymatic reaction occur on the cell surface, thus greatly improving the reaction efficiency. it can also avoid the degradation of foreign proteins by enzymes in the host cells, and overcome the drawbacks such as the inability of certain reaction products to carry out specific configuration folding in the cells. Bacterial cell display technology has been widely used in the screening of recombinant bacterial vaccines, whole cell biocatalysts, whole cell biosorbents and peptide libraries. It is widely used in environmental protection, microbial fermentation and other fields. This is also a good biotechnology in iGEM competition.
We think this can help future iGEM teams to think about whether the method they choose is appropriate, what are the advantages and disadvantages, and provide a new idea before designing experiments.
As a new type of prebiotic, the beneficial effect of chitosan oligosaccharide on human body is closely related to intestinal microorganisms. Chitooligosaccharides are widely used in the medical field, such as inhibiting cancer cells, reducing blood sugar, enhancing bone strength and promoting growth. Current studies have shown that chitooligosaccharides have inhibitory effects on a variety of cancer cells, such as liver cancer cell Bel7402, Lewis lung cancer cell, Hela cervical cancer cell line and human leukemia K562 cell line. Chitosan oligosaccharide has the ability of scavenging free radicals, which makes chitosan oligosaccharide have the ability to reduce blood lipids in diabetic patients and improve the antioxidant capacity of the body. Through the study on the expression of alkaline phosphatase gene in osteoblasts, it was found that chitosan oligosaccharide could inhibit osteoclasts and enhance the activity of osteoblasts, so that it could promote fracture healing and prevent the decrease of bone density. The free amino group in chitosan oligosaccharide molecule is a positively charged ionic compound, which can promote the proliferation of bifidobacteria in the intestinal tract and inhibit harmful bacteria. Compared with other chemical pesticides, chitosan oligosaccharide, as a biological pesticide, has the advantages of easy degradation, non-toxic and zero environmental pollution. Chitosan oligosaccharide not only has good efficacy, but also has the same dual biological regulation function as fertilizer. It was found that chitooligosaccharide could induce and activate plant immune system, improve plant antiviral ability, and significantly improve the quality and yield of agricultural products. The application of chitosan oligosaccharide, a bioactive substance, has been constantly updated and expanded in various fields. These literature information will help other teams to further understand the function of chitosan oligosaccharide which might be their final product.
Chitooligosaccharide is conjugated with gallic acid to prepare derivative of chitooligosaccharide (G-chitooligosaccharide). Research shows that it has an effect on free radicals in cells. Gallic acid and chitooligosaccharide are dissolved in methanol to form precipitation and store freeze-dried G-chitooligosaccharide. It is found to be an effective free radical scavenger, which can prevent oxidative damage to DNA, The level of cellular biomolecules and intracellular antioxidants can be increased by increasing antioxidant enzymes in living cells. Phenolic acid conjugated chitosan oligosaccharides have strong antioxidant activity, and acid acids such as p-coumaric acid, p-hydroxybenzoic acid and protocatechuic acid can also be used to synthesize these derivatives.
The bacterial display system cannot correctly express and fold the eukaryotic protein, and the eukaryotic yeast cell display system provides the possibility to successfully display the eukaryotic protein. Yeast is an eukaryotic, single celled microorganism whose cell wall is mainly composed of mannoprotein and β- Glucan composition. Glycosylphosphatidylinositol (GPI) anchored proteins have been widely used for the display of target proteins on the surface of yeast cells, such as mannose proteins that can extract glucanase, including lectins and flocculent proteins. Compared with phage display and bacterial surface display, yeast cell surface display has advantages in safety and genetic engineering. Yeast cells are widely used in food and pharmaceutical industries due to their safety. In addition, yeast cells are eukaryotic cells, allowing true folding, disulfide bond isomerization and glycosylation of expressed homologous proteins, especially complex eukaryotic proteins.
In terms of team building, we have the following suggestions for future iGEM teams:
1. Before forming a team, the team leader shall first determine the number of team members which should be
suitable for his/her research topic.
2. After the dry and wet teams are divided, there should be a clear division of labor, and the tasks should be assigned to everyone.
3. The team leader can assign work according to the strengths and advantages of each team member, so that the team members can actively complete the task.
4. The team leader needs to know the official deadline of each task and organize activities in an orderly manner.
5. During the experimental phase, team members must strictly comply with the requirements of the laboratory to ensure their own safety.
6. Due to the epidemic situation, please pay attention to epidemic prevention requirements when conducting offline activities.
7. In the experimental stage, you can record and update your notes at any time, so that the notebook and the engineering parts can be completed better when you make the Wiki.
8. After completing the experiment, please keep an eye on the iGEM official website to ensure the progress of the whole team.
9. Emphasize the team objectives, set the task deadline, and the team should hold regular group meetings to report the results of each team member.
We have held many lectures for students in Grade 10 and Grade 11 in Beijing and Qingdao who are interested in iGEM and biochemistry, to popularize their basic knowledge of synthetic biology, so as to help students who are preparing to create iGEM teams in the future accumulate background knowledge and ideas and precautions about experiments. In addition, we have established a public account to publicize our research topics and the official information of the iGEM competition, so as to help more people in need understand the registration process and stage tasks of the competition, and share our competition experience with future iGEM team members.