Project preparation and recruit team members

On January 12th, our team recruited members with various professional backgrounds in Hubei University of Technology (HBUT). The team members had a brainstorming session to discuss and specify the critical question that our project trying to figure out. Finally, we want to externally express a high value-added product. Our team members have conducted extensive literature reading research to find a suitable product in two weeks.

Finally, team leader and group leaders consulted to Dr. Yao and Liu (Associated professor in HBUT, PI of our team), and initially determined to make one of the exogenous expression of P37 family peptides for type 2 diabetes (T2D) therapy.

Modeling

1.Firstly, our bioinformatic group try to find out all the homological peptides derived from plants, and we utilized the Blast function in the NCBI database to search for protein sequences with high similarity to the Vg general sequence in the database. We obtained 50 proteins with a similarity higher than 71.3% to Vg from all species and removed 17 duplicate records. These 33 protein sequences were then aligned using the Align by ClustalW function in the MEGAX.(Figure 1)

Figure 1. The conserved regions of Vg evolution.Utilized the align by clustalw function of megax software, where the yellow regions represent conserved domains, and the white areas indicate mutation sites.

2.Using the MEGAX software, we constructed the corresponding phylogenetic tree by employing the Maximum Likelihood algorithm with the Jones-Taylor-Thornton (JTT) model.(Figure 2)

Figure 2. Phylogenetic tree of all the selected peptide.

3. Use the online tool cnsknowall (https://cnsknowall.com/) to optimize the phylogenetic tree. (Figure 3)

Figure 3. Phylogenetic tree of all the selected peptide.The data in this section is sourced from the blast in the NCBI database, employing the maximum likelihood algorithm of the Jones-Taylor-Thornton (JTT) model in megax software to construct a phylogenetic tree, which was beautified using cnsknowall (https://cnsknowall.com/). The red star marks Vg, the species in the green branches belong to Fabaceae, and those in the blue branches belong to Euphorbiaceae. No protein sequences with high overall similarity were found in animals and microorganisms.

Modeling

Our team utilized the deep learning architecture and neural networks of AlphaFold3 to predict the three-dimensional structures of Vg and its receptor. To better visualize these structures, we employed pymol for rendering. We observed disulfide bonds between the amino acids at positions 7 and 22, 3 and 15, and 20 and 32. (Figure 4)

Figure 4. Three-dimensional structures of Vg.(A) Vg has a spherical structure, which provides a basis for its oral availability. (B) it can be observed that there are disulfide bonds between the amino acids at positions 7 and 22, 3 and 15, and 20 and 32, which are crucial for the structural stability of Vg.

By using the latest Jpred4 algorithm in the JNet algorithm series, we predicted the secondary structure of VG. Utilizing the branch-site algorithm of PAML, we identified positive selection sites. In the NCBI database, we used the Conserved Domain Database (CDD) and tools such as BLAST (Basic Local Alignment Search Tool) and CD-Search to find a highly reliable Albumin. I superfamily functional domain in VG. By integrating the evolutionary strategies of different amino acids at positions 25, 27, and 28 in the 33 similar peptides, we finally formed a total of 26 mutants, which we named（R25A，R25A_V27F，R25A_V27F_V28A, R25A_V27F_V28I, R25A_V27L, R25A_V27L_V28A, R25A_V27L-V28I, R25A_V28A,R25A_V28I, R25G, R25G_V27F, R25G_V27F_V28A, R25G_V27F_V28I, R25GV27L, R25G_V27L_V28A, R25G_V27L_V28I, R25G_V28A,R25G_V28I, V27F, V27F_V28A, V27F_V28I, V27L, V27L_V28A, V27L_V28I, V28A, V28I). (Figure 5-7)

Figure 5. Utilizing machine learning to predict the secondary structure of Vg.The JPred4 algorithm exports predicted secondary structures of Vg, where the green regions indicate β-fold regions, with β-fold regions present at positions 21-31.

Figure 6. The branch-site algorithm of PYMAL.

Figure 7. Using machine learning to predict the functional domain determination of Vg.The conserved domain database (CDD) from the NCBI database was used, and through CD-search, a highly reliable albumin-I superfamily functional domain of Vg was identified. According to the annotations from the conserved domain database (CDD), the albumin-I superfamily functional domain can bind to the 43 kDa 3 eceptor protein VDAC-1.

Project · preparation

We held regular iGEM discussions to refine the design of the project. At the meeting, each of us was assigned corresponding tasks and each performed his or her own duties.

Modeling

To evaluate the binding potential of small molecule ligands, we used the chemScore to assess the activity, with results exported within a range of 0-1. The Hex scoring function and Ligand Docking were utilized to obtain Glide Score data, which provided insights into the binding free energy. For ease of data visualization and processing, we artificially converted all the binding free energy values to their negative equivalents, concentrating all the values into the first quadrant. We then used the Origin software to plot a three-dimensional graph representing these data. (Figure 8).

Figure 8. Using three abbreviated algorithms to evaluate the binding of Vg to the receptor VDAC-1.The x-axis represents the results derived from the chemscore algorithm in Maestro software, the y-axis represents the hex scoring function from Discovery Studio software, and the z-axis represents the glide score results from Maestro software. The binding site of VDAC-1 with Vg was again determined based on the binding positions obtained from the autodocking blind docking algorithm. In the figure, vdac-1 is displayed in stick form, the binding sites are shown as white surfaces, and the binding pockets are represented as purple cubes. The red spheres indicate Vg, while the blue spheres represent the other 32 evolutionary strategies. In the data processing, since the chemscore results are within the range of (0-1), and both the hex scoring function and glide score represent binding free energy, we artificially took the negative values of all binding free energy data for easier visualization and observation of conclusions.

Project preparation

Through the results of model, the teacher's suggestion and the current social needs, we decided to biological synthesize the hypoglycemic peptide (Vglycin, Vg) in Pichia pastoris.

Safety training

Before we start our wet experiment, all of our team members were subjected to the safety training by the lab manager, including personal protective equipment (PPE), the standard operating procedure of instrument, the physical biosecurity, information security and cybersecurity. Team members were required to pass certain exams before entering the laboratory for experiments.

Experiment

1.We designed the first plasmid, pPIC9K-His-DDDDK-Vg (Figure 9). Pichia pastoris was chosen as the expression system because it is a kind of methanotrophic yeast. We will send the designed plasmid to the company for synthesis, and start our experiment after getting the plasmid.

Figure 9. The construction of the plasmid pPIC9K-His-DDDDK-Vg.

2.Plasmid pPIC9K-His-DDDDK-Vg was transformed into Pichia pastoris GS115. We performed single enzyme digestion, linearization, agarose gel electrophoresis verification and DNA purification of the newly constructed plasmids. PCR amplification and electrophoresis verification. (Figure 10)

Figure 10. Coated plate sieve.

3.Bands were observed on SDS-PAGE through Western Blot experiment. But the molecular weight didn't match our expectations.

4.We conducted nickel column affinity chromatography to isolate the target protein with His label, and found that there were no bands.(Figure 11)

Figure 11. The expression of His-Vg detected by western blotting.

Modeling

Using the Vina force field in conjunction with the Autodocking algorithm, as well as the OPLS-AA and AMBER force fields, we conducted calculations with all three methods employing the TIP4P water model and the solvent models GBSA (Generalized Born Solvent Accessible model) and MMGBSA (Molecular Mechanics Generalized Born Surface Area) algorithm. To integrate the results from these three algorithms, we used Origin software to plot a three-dimensional graph. To maintain consistency in our data representation, we also converted all the binding free energy values to their negative equivalents, ensuring that all the values were concentrated in the first quadrant of the graph.

Experiment

1.As for the problem of the first plasmid we constructed, we speculated through literature review that the signal peptide in the front end covered up the expression of His-tag, resulting in the failure of the recombinant protein to hang on the nickel column. Therefore, we replaced DDDDK with Asp-Pro to improve the expression of target protein.

2.Therefore, we optimized the plasmid to obtain the second plasmid, pPIC9K-3Vg-His. (Figure 12)

Figure 12. The construction of the plasmid pPIC9K-3Vg-His.

3.After designing the second plasmid, we send it to the company for synthesis.

4.After getting the constructed plasmid, we carried out experiments, including plasmid extraction, linearization, DNA purification, PCR amplification and nucleic acid gel electrophoresis.

5.However, in the final western blotting experiment, we did not detect expected bands by the western blotting. (Figure 13)

Figure 13. The expression of Vg-His was detected by western blotting.

Human practices

In order to understand the current domestic hypoglycemic drug market and diabetic patients' expectations for hypoglycemic drugs, we came to the retired staff activity Center to conduct voluntary blood glucose screening for retired staff. (Figure 14)

Figure 14. Blood glucose testing for elderly people in the community.

Experiment

1.On the basis of previous experiments, we considered that the low molecular weight of the recombinant protein made it difficult to be detected.

2.Therefore, we added a nattokinase NK in front of Vg, and designed the third plasmid pPIC9K-NK-3Vg-His (Figure 15), and sent it to the company for synthesis.

Figure 15. Construction of the plasmid pPIC9K-NK-3Vg-His.

3.Through the experimental operation, we carried out plasmid extraction, linearization, DNA purification, PCR amplification, nucleic acid gel electrophoresis. Finally, we make experimental results and successfully detect bands on SDS-PAGE.

4.The above experiment is repeated, and the protein we need can still be detected. (Figure 16)

Figure 16. The expression of His-tag target protein was detected by western blotting.

Human practices

1.Our team members visited BGI and heard that BGI's gene therapy program for chronic diseases is to treat diabetes by adjusting the nutritional structure of food and adding b vitamins. We also reported our work to BGI, and one of our strengths- the homology of medicine/food, which coincided with BGI's program and it was highly praised by Chen Liu, an expert from BGI. (Figure 17)

Figure 17. Presenting our project to BGI.

2.Our team members participated in the 11th iGEMer Exchange Meeting (CCiC) in China. At the meeting, teacher Ziwen Xie put forward a valuable suggestion that our project needs an independent innovation iteration process. (Figure 18)

Figure 18. Participating in the 11th iGEMer Exchange Meeting in China (CCiC).

3.To further explore the latent value of the existing achievements, our team selected the backbones to establish an entrepreneurial team. We are guided by the spirit of iGEM to make real and inexpensive hypoglycemic drugs for more than 140 million diabetic patients in our country. 

Experiment

1.Due to the successful expression of the protein, we enlarged our expression system (200 mL of LB) to improve Vg yield (Figure 19).

Figure 19. Vg expression induced by methanol in Pichia pastoris.

2.To further increase the stability of Vg, we redesigned our construction of Vg expression system. By reviewing the literature, we found that the stability of disulfide bonds can be maintained by adding molecular chaperones. We hope that by adding molecular chaperones, such as Asf1, we can help boost the activity of DNA helicase during cell division.

3.At the same time, enhancers such as heptapeptide Qa, which has been reported to enhances viral protein expression, was added to increase the expression of recombinant protein.

Human practices

1.Our instructor Dr. Chenguang Yao introduced us to Ms. Hongrong Liu, the boss of Wuhan Lanzhi Biotechnology Co., Ltd. Our team immediately rushed to Wuhan Lanzhi Biotechnology Co., Ltd. We emphasized that we have determined the unique certainty of drug ingredients, the homology of medicine and food, and the advantages of increasing production capacity by using synthetic biology. I received enthusiastic support from Ms. Hongrong Liu and her company (Figure 20).

Figure 20. Visiting and communication with Wuhan Lanzhi Biotechnology Co., Ltd.

2.We interviewed Kanghong Hu, the director of the Sino-German Biomedical Center of Hubei University of Technology. We asked some questions and received some useful advice from Professor Hu (Figure 21).

Figure 21. Interview with Professor Kanghong Hu.

3.Our team also participated in the synthetic biology competition held in China and won the silver prize (Figure 22).

Figure 22. Our team won the silver Prize in Chinese Synthetic Biology Competition.

Modeling

Using the Vina force field in conjunction with the Autodocking algorithm, as well as the OPLS-AA and AMBER force fields, we conducted calculations with all three methods employing the TIP4P water model and the solvent models GBSA (Generalized Born Solvent Accessible model) and MMGBSA (Molecular Mechanics Generalized Born Surface Area) algorithm. To integrate the results from these three algorithms, we used Origin software to plot a three-dimensional graph. To maintain consistency in our data representation, we also converted all the binding free energy values to their negative equivalents, ensuring that all the values were concentrated in the first quadrant of the graph. (Figure 23).

Figure 23. Further, using three precise algorithms to evaluate the binding of Vg to the receptor VDAC-1. The x-axis represents the results derived from the vina force field combined with the autodocking algorithm, the y-axis represents the results from the mmgbsa calculation scheme using the opls-aa force field, and the z-axis represents the results from the mmgbsa calculation scheme using the amber force field. The red spheres indicate Vg. The orange spheres represent the top three mutation strategies in terms of binding effectiveness with vdac-1 excluding Vg. The blue spheres represent the other 29 mutation strategies.

Experiment

1.Through the construction of plasmids from generation to generation, the addition and updating of components, we finally successfully obtained the ideal band and realized the large-scale expression of hypoglycemic peptide P37 by Pichia pastoris.

2.We have sorted out the experiments during this period. Under the previous experimental results, the protein was purified by nickel column and concentrated. The purified protein was detected by western blotting assay.

Human practices

In order to popularize students' understanding of biology and synthetic biology, our team visited Wuhan Yucai Senior High School to introduce the concept of synthetic biology and its role in the pharmaceutical industry and in maintaining human health (Figure 24).

Figure 24. Popularizing students' understanding of biology and synthetic biology.