Team:Nanjing-China/Design

After putting forward polyphosphate as our main research object, we also dialectically found many problems of polyphosphate as a fireproof material. As an inorganic material, its main industrial synthesis method is chemical synthesis, in which many toxic gases will be produced. Therefore, in the exploration process of the project, through the experimental design of synthetic biology, we explored the pathway of biosynthesis by means of Citrobacter freundii. Compared with chemical synthesis, this biosynthesis method can fully utilize the advantages of engineering bacteria, which is significantly valuable with high efficiency, environment-friendliness, high yield as well as simplicity and convenience in condition control. This is also the first innovation point of our project.

In addition, following the idea of "novel biosynthesis organic-inorganic materials ", our team has achieved a pretty good combination of organic macromolecules and polyphosphate molecules with different properties through transformation, so that the two materials can respectively express their superior properties, and expand the material’s wearability and fluorescence. This is the second innovation point of our team.

The Very Start

First, we told our team instructor—Professor Wei Wei about our idea and asked for the direction of the experiment. Prof. Wei 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. Therefore, it is extremely challenging to select suitable materials.

Prof. Wei suggested that we can start from the combination of green fluorescent protein(GFP) and polyphosphate. According to Prof. Wei, 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.

Next, we interviewed Professor Zheng Peng from the School of Chemistry and Chemical Engineering of Nanjing University. Professor Zheng pointed out that polyphosphate should bind to proteins with more positive charges on the surface. He put forward suggestions with considerable instructive significance for our project. He believes that the fire resistance of our polyphosphates can be combined with wearability and is expected to be applied to fire-retardant and flame-retardant textile materials. Prof. Zheng also pointed out that for the binding effect between polyphosphate and spidroin, we should first observe the state of the solution after the two are mixed. He suggested that when we try to bind polyP and GFP, binding conditions should be found in the phase separation region where they form gel.

According to Mr. Zheng's suggestion, we investigated Loqi textile, hoping the idea of "organic-inorganic biological synthesis new material" can be recognized, in order to promote the application of synthetic biology to a broader prospect. The manager of the company spoke highly of our idea and pointed out that we could combine polyP with textile materials such as spidroin to obtain materials with better handle and toughness.

PolyP Pro

Based on our previous investigation and in-depth discussion, we started the experiment. We constructed the plasmid of + 36GFP, and connected the target gene sequence of + 36GFP to PET29a vector to obtain the recombinant plasmid PET29a - + 36GFP. After expression and purification, we explored the binding conditions under the proposal of Mr. Zheng, and achieved the stable combination of + 36GFP and polyP, obtaining expected good results.

Next, we carried out the combination of spidroin and polyphosphate. We first retrieved the amino acid sequence of the recombinant spidroin, and searched for its crystal structure on the PDB website, but the complete protein structure was not found on the website. Therefore, we conducted a hybrid modeling of the recombinant spidroin through a computer, and The Autodock molecular docking software was used to simulate the binding of recombinant spidroin and polyphosphate. Since polyphosphate has a large amount of negative charge, while the existing recombinant spidroin has only a small amount of positive charge under physiological pH conditions, we can see from the results of computer simulation that the binding energy of the two is higher, and the interaction is weak, such a direct combination is difficult to form the organic-inorganic hybrid material we expected. Therefore, in order to satisfy enough electrostatic interactions with polyphosphates, without affecting the overall structure of the recombinant spidroin, we put the N-terminal and C-terminal parts of the natural spidroin away from the key parts of the rigid structure. The amino acid was mutated into a positively charged amino acid, and an expression vector of the mutant recombinant spidroin was constructed, and the protein induced expression and purification experiment was performed.

Although the combination of sp & polyP is predicted to be stable, which indicates that our mutation of sp is a success theoretically, according to our modeling. However, in the process of protein separation and purification, most of the spidroins cannot be specifically adsorbed to the cation exchange resin. It flows out directly with the original protein solution, which makes it difficult to carry out subsequent elution steps. This has a great impact on the later hybridization step.

Improvement of our experiments

In response to the difficulties and problems that occurred during the experiment, our team sought advice from professional teachers.

We first paid a return visit to Professor Zheng Peng. He believes that the core reason for the difficulty in hanging the column is that the team has problems with the design of the spidroin. Before the three-dimensional structure of the spidroin is resolved, it is difficult for us to artificially design the charged condition of the protein. In the high-level structure, part of the charge is wrapped inside and not exposed on the surface of the protein, which makes it difficult to contribute to the charge distribution of the protein. The second is to investigate the combined structure of the two, characterize it through appropriate methods, and then determine its binding force through a series of methods.

Next, we visited the research group of Professor Cao Yi and Professor Tian Ye from Nanjing University. They provided our project with opinions in another direction from the perspective of the physical properties of biological materials. Prof. Cao pointed out that as a natural protein, spidroin has extremely high toughness and strength, which has a lot to do with its crystallinity and the regulation of its three-dimensional network structure. It is currently difficult to achieve a complete artificial simulation. For the process of hybridization with organic and inorganic materials, we have to consider the length of polyphosphate, spidroin structure, the amount of charge and the charge density, these factors have a greater impact on the ratio of the combination of the two. Prof. Cao thought some certain amino acids are largely needed in the synthesis of the spidroin, which might be the reason why we failed. For example, the glycines and alanines form β-plated sheets in the spidroin. Maybe some extra amino acids shall be added into the culture mediums. Their suggestions reminded us to consider completing protein purification and subsequent binding experiments by constructing +36GFP-spidroin fusion protein. And Prof. Tian stressed that the cations in the solution, namely the salt we add into the system, decide the shape of the polyphosphate chain. The polyphosphate chains will be extended in pure water, for the negative charges repel each other in a system with a low cation concentration. When the cation concentration increases, the polyphosphate chains begin to aggregate. Prof. Tian’s suggestions reminded us to use solution containing Na+ as cation exchange column buffer in the process of purification and use high concentration Na+ solution.

Finally, under the guidance of teachers and the efforts of students, the +36GFP-spidroin fusion protein was purified.