Team:Nanjing-China/Human Practices

Before our project began, a true story happened to our team member Zhongqi Xu attracted our attention to fire accidents. During a whale watching tour, he witnessed a fire accident in which not only did fierce smoke and burning flames devour a ship but also ignited the tarpaulin of Xu's boat.

Although there was no danger in the end, the boat fire was put out in time and no casualty were caused, this experience of escaping from fire left Xu a deep expression, and initiated our thinking about the fire.Fire, brings human light and energy, along with fear and disasters, smoke, flame, tears.

In the last decades, fire has become a world-wide crucial security issue.

As the saying goes, to forestall is better than to amend. On one hand, the development of reliable fire-retardant materials is the most valid weapon against fire. On the other hand, when firefighters rush into the fire rescue, they also need more protection and guard. Fire clothing, as the focus of continuous improvement in fire protection work, highly requires the development of lightweight, waterproof and breathable fireproof fabrics, which is a significant role that new materials can play for fire safety.

The research and development of fireproof materials has always been a hot spot in the research and development of new materials.In the early stages of the project (2019.12),Our team members investigated webpages of many fireproof material companies and interviewed the manager from Gesanghua Paint Company, hoping to get some help and guidance for the overall idea of the project.

Interview with Gesanghua Paint Company

Our team members conducted interviews on the topic of fire-retardant coatings with Mr. Da Jiang, the manager of Gesanghua Paint company. We widely consulted on the relevant aspects such as industry situation, professional knowledge, equipment technology and market prospect.

As for the production technology of fire-proof materials, Mr. Jiang mentioned that many innovative methods have been tried in the production process of his company. In order to realize fire-retardant functions, the use of multiple materials replace single raw materials. Therefore, the key problem lies in the proportion of mixed materials.

In terms of demand for fireproof materials, Mr. Jiang thinks that not only should they have certain fire resistance characteristics, but also they should possess excellent performance in corrosion resistance and strength. Moreover, the fireproof property of different material varies a lot. The fireproof level of the material often determines its application field and what kind of fire prevention task it could undertake. To sum up, different places often have diverse needs for fire-resistant materials. Therefore, the research and development of fire-proof materials has a long way to go and cannot be taken in the lump. At present, in this field, Mr. Jiang said that fireproof materials are still confronted with many problems that need to be solved urgently. There is a lot of room for adjustment and improvement in the process of production and use. This is also the vitality and core of the fire-proof material industry currently. How to develop fire-retardant materials with more new types, more powerful functions, more environmentally-friendly production process and more targeted application fields is the key issues, which is also Mr. Jiang's expectation as a industry veteran.

The results of the preliminary investigation show that traditional fire proof materials cannot meet the various needs of current market, and there are still many pain points that can be improved. At present, the best-selling fireproof materials on the market can be divided into water-based and solvent-based based on the solubility of the coating itself, and can be divided into intumescent and non-expandable based on the foaming performance of the coating. The most common fireproof coatings are mainly inorganic materials represented by calcium carbonate, vermiculite, perlite, etc., and coagulants-resins, flame retardants-ammonium polyphosphate, blowing agents-melamine, solvents-poly Pentyleneerythritol, etc. is used as a set of organic materials in a flame retardant system.

Traditional flame-retardant materials still have shortcomings and drawbacks that need to be resolved in many aspects:

On one hand, the durability of materials is also an issue that cannot be ignored. This material is often used on the surface of buildings, cables, clothing, etc., and the contact surface with the external environment is relatively large. The effects of natural weathering, exposure, rain erosion, and acid-base corrosion in special environments are extremely important for its fire resistance. test. At the same time, many human factors also affect its durability. For example, a fire-resistant garment needs to be able to maintain a certain degree of fire resistance even after repeated washings. If a material cannot achieve good durability, it is difficult to escape the fate of being eliminated.

On the other hand, the scalability of the material is not strong, and the application field is single. Some materials are relatively stable in nature, but have few surface active groups, or the material structure is difficult to modify... Various reasons make it difficult to properly combine with other materials, making them only applicable to the single field of fire retardant coatings , Can not properly combine with other organic biological macromolecules, the competitiveness of the material will be slightly inferior.

In addition to the above aspects, more superior flame retardancy and fire resistance, thermal stability, material strength and other aspects also need to be further strengthened. Therefore, it is urgent to develop novel fire proof materials. And we believe that our idea of “novel biosynthesis organic-inorganic materials” and the success of engineering and project will make the world a better place.

Our project is beneficial to the mankind

On the afternoon of October 24, 2020, we participated in the " Genscript & Nanjing's University：Synthetic Biology Discussion Salon" hosted by Genscript. At the salon, we discussed the biosafety and ethical issues related to our project with Genscript. After communication and discussion, we came to a conclusion that our project complies with safety regulations and is beneficial to the whole world and mankind.

Use technology of synthetic biology to achieve pollution-free production process

In addition to the drawbacks mentioned above, traditional fire proof materials also have another shortcoming according to the interview with Gesanghua Paint Company. The most critical issue is the environmental problems and pollution of materials. The solvents involved in the production and processing of solvent-based fire-retardant coatings are mainly flammable and explosive liquids containing benzene and alcohols. At the same time, they have certain toxicity and high carcinogenicity. They are the main pollutants in the production and use of coatings. Dust substances and volatile gases involved in fire retardant coatings are another important issue to be faced. Many inorganic materials can cause incalculable pollution if they cannot be properly sealed.

As far as we know, polyphosphate as an inorganic material, is mainly industrially produced by chemical synthesis, which will produce a lot of toxic gases. In our project, through the experimental design of synthetic biology, we explored a pathway of biosynthesis of polyphosphate using Citrobacter freundii. Compared with chemical synthesis, this method of total biosynthesis can fully use the advantages of engineering bacteria, with high efficiency, environmental friendliness, high yield and simplicity and convenience in condition control. Based on our interview and investigation, we determined that the concept and method proposed by us can fill the gap in the manufacturing process of existing fire-retardant materials.

Explore the unknown and promote scientific development

According to our previous research, we know that polyphosphate is widely used in chemical synthesis, but its application in biology still needs more exploration. Actually, polyphosphate may have hemostatic effect in animals, while its antagonists may act as antithrombotic / antishock agents. It has been found that the addition of polyphosphate can alleviate the decline of plasma coagulation function of platelets in patients with Hermansky Pudlak syndrome. In animal cells, only bone tissue cells need polyphosphate as a storage tool. Vertebrate bones are mainly composed of a calcium phosphate called apatite. In plants, polyphosphate can be used as a heavy metal chelator. Expressing bacterial polyphosphate kinase produces a large amount of polyphosphate in transgenic tobacco to resist the effects of heavy metals such as Hg2 + on plants. In microorganisms, polyphosphate plays a key role in the process of stress resistance, colonization and infection. Long chain polyphosphate can also lead to bacterial growth inhibition, pathological changes and Bacillus cereus cracking. In the industrial field, commercial ammonium polyphosphate is often a mixture of orthophosphate and polyphosphate. Polyphosphate has been used in plant phosphate fertilizer and food additives. In addition, ammonium polyphosphate can be used as a halogen-free, efficient and non-toxic flame-retardant material, which is the focus of our project. We hope to combine it with biological macromolecules with rich biological functions so as to realize the production of novel organic-inorganic hybrid materials. To realize our idea, we further consulted our instructor, Professor Wei Wei.

Prof. Wei spoke highly of our idea, but he also said that the combination of organic and inorganic materials is currently a frontier research field, and related research is still in the exploratory stage. There are currently no mature examples for reference and learning in the field. The combination effect of most organic-inorganic hybrid biomaterials depends on specific experimental results and is difficult to predict theoretically.

We believe that the success of our project will expand the application field of polyphosphate, showing its marvelous performance in the field of synthetic biology. We also hope that the success of the project can promote the research process of organic-inorganic hybrid materials, and our new method of using synthetic biology to realize hybridization could provide new insights for researchers in related fields around the world.

Our activities impacted our project

Professor Wei Wei: Verify your ideas starting with GFP

After determining the idea of our project, we had an in-depth discussion and communication with our project instructor Professor Wei in January, 2020.According to Prof. Wei, though our idea should be encouraged, it is extremely challenging to select suitable materials. He then suggested that we can start from the combination of green fluorescent protein(GFP) and polyphosphate. GFP has the characteristics of fluorescence and strong positive charge. It has a considerable prospect, high operability and implement ability, and can be used as a direction for our early exploration, both in terms of the difficulty of binding and the characterization of the binding result.

We accepted Professor Wei's suggestion. At the beginning of the experiment, we constructed a strong positive GFP protein: + 36GFP. Meanwhile, in order to better simulate the combination of polyphosphate and protein, our modeling group modeled the three-dimensional structure of protein according to the sequence information provided by the experiment group.

Professor Wei’s suggestion helped us to carry forward our project from idea to lab. According to his proposal, we have a preliminary idea for the realization of the project.

Professor Peng Zheng: Use electrostatic force, find the phase separation region of gel state

At the beginning of the experiment of our project, the most critical problem that our experiment faced to be solved urgently was how to combine polyphosphate and protein. With such doubts and confusion, we interviewed Professor Zheng Peng in the winter vacation at the beginning of 2020.

Interview with Prof. Zheng

Professor Peng Zheng is an outstanding scholar in the field of organic-inorganic hybrid materials and protein biomacromolecules. Professor Zheng pointed out that polyphosphates, as inorganic linear materials with strong negative charges, should be bound to proteins with more positive charges on the surface. Such electrostatic binding is a possible direction of this project. Based on years of research experience, Professor Zheng said that for the binding effect between polyphosphate and protein, we should first observe the state of the solution after the two are mixed. When the two are combined in an appropriate proportion, the colloidal solution formed is a sign of the combination. Secondly, the structure of the combination should be tested, and then characterized by appropriate methods. We should also test the binding force using series of methods.

We accepted Professor Zheng's suggestion, and began to consult the relevant literature after receiving the plasmid of +36GFP protein. Finally, we found the suitable binding conditions in the gel phase separation region by electrostatic effect. According to this idea, the modeling group also simulated the binding of protein and polyphosphate. Professor Zheng's suggestion runs through our whole project and provides valuable suggestions for us to explore the combination of spidroin and polyphosphate. During the interview with Professor Zheng, we gradually have a clear understanding of how to realize our project.

After the interview with Professor Zheng Peng, the rest of our HP project was planned to continue after the Lunar New Year. Unfortunately, COVID-19 broke out in late January 2020, and our project was temporarily suspended due to the outbreak of the virus.From February to June, due to the epidemic prevention and control requirements, we had to isolate ourselves at home and were not allowed to return to school, so our social practice was difficult to carry out offline.However, our team did not give up on speaking out of turn. During this period, our team members made active use of network resources and carried out several online activities, including the publicity activities of synthetic biology and the core spirit of IGEM contest with the help of the school's recruitment platform, Professor interviews on open weekends and so on.

Around July, due to the effective control of the epidemic, our team members were able to return to school and continue to carry out social practice activities.

Leqi Textile: Combine spidroin with polyphosphate

At the time when the combination of + 36GFP and polyphosphate is about to succeed, we interviewed Leqi Textile in the summer after returning to school to broaden the application field of our "organic-inorganic biosynthesis new materials"

Interview with Leqi textile

Yixing Leqi Textile as a well-known local dyeing and textile enterprise in Jiangsu Province has a high reputation and recognition in the industry. Our interview mainly focused on the clothing production, textile technology and fire-proof clothing related to the company's production and operation field. We also consulted the general manager of the company, Qiaoyuan Zhen, on the industry's relevant progress, professional knowledge, professional knowledge and requirements as well as market demand.

During the interview, Mr. Zhen affirmed our idea of new textile materials. After learning about our experiments, he suggested that we could try to combine polyphosphate with spidroin, a new textile material with high toughness and strength. He believes it could be a promising new material for organic-inorganic biosynthesis. For the application prospect and market demand of the project, our team members further contacted relevant companies and enterprises, extensively investigating the current market situation. When interviewing the manager of Leqi Textile company, we learned about the existing fabric manufacturing process and fabric yarn requirements, among which strength, toughness and evenness are most important and stressed. In addition, the development and production of fireproof fabrics have great potential and market in China and even in the world. The current cutting-edge technology of fireproof fabrics is still foreign patents, which still have disadvantages such as pollution in manufacturing process and insufficient comfort. Therefore, manager Zhen expressed strong expectation for the final products of our project.

We accepted the suggestion of manager Zhen. Since the natural spidroin is relatively negative charged, making it stably combine with polyphosphate is theoretically impossible (it is not denied that it can bind to some extent), so we first retrieved the amino acid sequence of the recombinant spidroin. Similarly, as a new protein, PDB can not find any 3D structure information about it. Therefore, our modeling group predicted the structure of modified spidroin and simulated its binding with polyphosphate.

Leqi textile's proposal highly promoted the progress of our project, enabling us to apply "novel materials for organic-inorganic biosynthesis" to a wider range of fields. We are also prompted to start improving the theory of stable combination of organic and inorganic molecules by electrostatic interaction. After an interview with manager Zhen, we have a clearer understanding of the future of our project. In a sense, this interview has helped us open the door from the laboratory to the market and even the whole society.

Professor Yi Cao: Combine positive charged GFP with Spridroin

In the middle of the project, when we went back to school after the outbreak was under control, we ran into a bottleneck in trying to purify and modify the electrical properties of spider silk proteins. At this time, we contacted Professor Cao Yi and did an interview.

Interview with Prof. Cao

Professor Yi Cao 's research field is mainly synthetic protein materials. He gives us relevant suggestions from the perspective of spidroin design. For the combination of polyphosphate and spidroin, we should reasonably transform the original natural negatively charged spidroin. Professor Cao pointed out that in the process of transformation, we should first consider the length of polyp, the length of spidroin structure, the amount of charge and the charge density. These factors have a great influence on the combining ratio of the two. It shouldn’t be modified at will, especially shouldn’t destroy the key nucleotide sequence of the structure determining site of spidroin. Secondly, the folding of spidroin also has a certain impact on the electrical modification. If the modified positive charge is wrapped in the structure, it will play a small contribution to the electrostatic effect of binding, where a good binding remains difficult. For spidroin, a protein whose three-dimensional structure has not been fully analyzed, it is difficult to reconstruct its electrical properties.

Our team members also reported the results of our previous experiments to Professor Cao, based on which Professor Cao Yi proposed a new idea for us. He thinks we may consider assembling spidroin with GFP protein which has successfully achieved the binding, and then use GFP as the intermediary to connect polyphosphate and spidroin. At the same time, the fluorescence of GFP can easily show and track the results of experiment, which is highly feasible and innovative.

Finally, Professor Yi Cao explained in detail the relationship between the structure and function of spidroin and the spinning process of spidroin in animals. He also explained and the regulation of crystallinity between different regions and the control of the newly formed three-dimensional structure network, which is key to its high tenacity and high strength. Meanwhile, he communicated with us about the bottleneck of spidroin research in the field, the application of modified materials and its broad prospects. In all professor Cao expressed his high expectation for our project.

Professor Cao gave some valuable information and suggestions. This interview inspired us to combine spidroin with + 36GFP we made to form a new fusion protein, and then combine the fusion protein with polyphosphate.

This interview has opened up our mind and pointed out another way for our project, helping us overcome one of the biggest difficulties in this project.

Professor Ye Tian: Use Sodium ion solution as buffer and eluent

At the same time, we interviewed professor Ye Tian.

Interview with Prof. Tian

In the process of protein purification, we also encountered a problem that it is difficult to purify and separate the modified protein. For such a problem, we interviewed professor Ye Tian from the Department of modern engineering and applied sciences. Professor Tian showed a strong interest in our experiment progress and provided his professional knowledge to help us with our confusion. He thinks that the selection of buffer is very important for protein elution. In order to maintain the biological activity of protein, it is often eluted in salt solution with different salt concentration. For the selection of buffer salt ion, we usually use monovalent sodium ion or bivalent magnesium ion salt as buffer to elute positively charged protein. Professor Tian said that we can use different concentrations of sodium chloride solution gradient elution and conduct parallel experiment to explore the best separation conditions.

Professor Tian also provided a good suggestion for our bottleneck problem. We used the appropriate buffer and eluent according to his suggestion, and successfully completed the purification of the fusion protein.

Summary

At the end of this season's competition, we interviewed again with Leqi Textile and Gesanghua Paint Company, where we received a high appraisal and good feedback. They agreed on this novel material's great significance for the prevention and control of environmental pollution and social life security. The application potential of the material was highly recognized for its breakthrough from traditional polluting production to contamination-free process of biosynthesis. Generally speaking, all innovation of our project have been affirmed and recognized by our interviewees.

To sum up, our project is an in-depth study on the innovation and improvement of fireproof materials, which is a hot issue in the current social life and fire-fighting work. Through the application of the concept and knowledge of synthetic biology, we can design a product with good thermal stability, pollution-free production process, high biocompatibility, good wearability, strength and toughness and finally develop a new type of organic-inorganic hybrid material with excellent properties. In the design process of the material, we fully realized the experiment idea of social practice oriented. HP group conducted diversified research: In terms of market prospects, we interviewed a number of enterprises and companies, conducted online research on several enterprise websites and inspected the terminal and application of the material. In the process of laboratory exploration, we consulted several professional professors, widely absorbed and adopted the guidance suggestions and ideas. From the experimental design requirements to market application requirements, the detailed guidance and feedback of the experiment process are carried out in many aspects, which is an important force to promote our experiment progress. Finally, based on comprehensive opinions of many aspects on our experiment design products, we can draw the following conclusions: The material is of great significance for the prevention and control of environmental pollution and social life security. The application potential of the material lies in the breakthrough from traditional production that cause a lot of pollutant emissions to pollution-free process of biosynthesis. Organic materials are hybridized to improve and optimize the performance of inorganic materials with excellent properties, and facilitate the innovation of material synthesis. Finally, all innovation of our project have been affirmed and recognized by our interviewees.