Team:BNDS China/Poster

Poster: BNDS_China

Cross-linking Bacteria Cellulose with Recombinant Structural Protein

Poster presented by: BNDS_China
Authors and their Affiliated Institution: Wanji Li, Chonye Han, Lixing Wang; Jingcheng Yu, Haoxuan Yin, Nuo Shi, Ruorui Zhang, Lihan Lin, Yuyue Jiang, Yijia Zhang, Qiyu Fu, Yiqi Chen, Yuyan Chen, Xinyu Zhang, Wenfei Xiao, Xiaoda Zhang, Keru Tang, Shuyan Wu from BNDS

Abstract

Wild animal’s leathers have great values for the clothing and fashion industry. Some luxury products like crocodile bag even require leather from rare species. This profitable business drives poachers to illegally hunt and hurt those wild animals. The demands of leather put the wild animals in a dangerous place and the harms caused by poaching for species conservation is unthinkable. Therefore, we aimed to integrate bacterial cellulose and protein crosslinking to manufacture artificial leather for animal conservation. Specifically, we utilized G. xylinus to synthesize bacterial cellulose. Moreover, we engineered E. coli and P. Pastoris to purify collagen-like protein and spider fibroin with SpyCatcher003 and SpyTag003 for protein crosslinking. This synthetic biological method provides an alternative way to supply raw material for leather industry, while ensuring animal and environment conversation.

INTRODUCTION

As people’s consumption of animals’ leather rises and the pollution brought by current artificial leather processing and disposal becomes more and more serious, it is necessary and urgent to find a solution for the conservation of animal and environment. Thus, BNDS_China aims to find a both biologically and environmentally friendly alternative of natural leather. After researching lots of eco-friendly materials, what we have decided to use to make artificial leather are bacterial cellulose. By using bacterial cellulose as backbone, using different proteins as modifications and using the Spy Catcher and Spy Tag technology as tanning agents, we are able to fulfill all the concerns of our artificial leather’s tenacity, elasticity, abrasion resistance, texture and overall structural strength.

INSPIRATION

Wild animal’s leathers have great values for the clothing and fashion industry. This profitable business drives poachers to illegally hunt and hurt those wild animals. The demands of leather put the wild animals in a dangerous place and the harms caused by poaching for species conservation is unthinkable. So, at this point, the need for leather alternatives is crucial.

Thus, we, BNDS_China, aimed to design and manufacture a brand-new artificial leather as an alternative regarding the leather supply shortage. We planned to design our artificial leather based on the bacterial cellulose as the backbone and the crosslinked structural protein network as additives. In this way, a piece of artificial leather with great structural property would be made.

MODELING

Why Model

In iGEM 2021 BNDS_China, we utilized the latest version of SpyTag and SpyCatcher: SpyTag003 and SpyCatcher003 and make them to connect to each other in an optimal way such that the products' viscosity and strength would be close to a real leather.

Mechanism

The ratio of the reactants: SpyTag and SpyCatcher is found to be an influential factor of the reaction. Then our goal of reaching the gel point can be interpreted as the inequality with the amount of SpyTag and SpyCatcher as independent variable.

Fig1. The graph colored as yellow represent C(Na,Nb). The graph colored as blue represents the plane whose value is zero.

By solving the critical conditions, two functions of the critical ratio, namely the boundary of the range of ratio of SpyTag003 and SpyCatcher003 when gelation occurs, are obtained, where x is the sum of amount of SpyTag003 and SpyCatcher003.

Result

By plotting, we found that SpyTag003 and SpyCatcher003 formed the lower and upper critical boundaries of the gel, both around 0.8. Therefore, we can get the optimal ratio of SpyTag003 and SpyCatcher003 is 0.8.

FUTURE DIRECTION

In the future, several potential field could be developed in order to perfect our product. The purpose of envisioning is to improve our project to be more customizable and commercial, creating extra value in the society. Depending on customers who would be interested in our leather, there are two relative direction of improvement.

End-users:

people who want to experience “leathers” with enhanced mechanical properties and care about wild lives and its protection.

Vision 1:

Manufacturing our products that have different colors and figures using light-controlled pattern of coloration.

Reason: The light-controlled technique is accurate and momentary, since it avoids the delay and diffusion of chemical inducement.

Vision 2:

Applying in various situations.

Reason: Combination of those two great mechanisms, numerous products with leather-like endurance and touch as well as 100% customizable patterns can be used in areas of clothing, packaging, and even medical fields. From making a luxury but eco-friendly handbag with decorative dying on it to a surgery mask made from bacterial cellulose and recombinant structural proteins, all of these potential fields are possible for our artificial leather.

So Long As We Are Imaginative, Nothing Is Impossible.

Safety

Another important factor to emphasize is the safety problems of our project. On the one hand, all of our products should be completely sterilized and purified to remove endotoxin before getting out of our lab. In addition, in our project, chassis organisms like P. Pastoris, E. coli, and G. xylinus are all at biosafety level 1, which would not cause any biosafety issue.

Integrated HP

Beer Yeast

Because we are going to make leather from bacterial cellulose, there will be a large-scale fermentation. After the research about the percent yield of bacterial cellulose made by different materials, we found that our program can use beer yeast as the raw materials to produce bacterial cellulose, which is also environmental-friendly.

Then we came up with the idea of creating a new process to recycle the beer residues. We did an interview about beer residues in a home-brewing bar in 798-art center in Beijing.

After interviewing the professional, we learned that making beer will produce many residues such as malt and yeast. Malt are usually considered as kitchen waste and directly thrown away, while yeast carcasses are sticky, so they usually flushed straight down the drain. So, if we can recycle these materials, they cannot only contribute our experiment, but also protect the environment.

Ancient Papermaking

As our desired product for the project is to produce bacterial-cellulose. We have related the unshaped nature of the cellulose production to the crafts in ancient papermaking. Transforming woody bast fibers into handmade papers through a series of technical flow, ancient papermaking is turning the intangible pulp into the various shaping of paper.

Luckily, we contacted the expert of ancient papermaking Mr. Gong Bin to be our interviewee. During the interview, Mr. Gong demonstrated us his workshop with many processing papers along with tools and materials. Mr. Gong also shared with us the general process of ancient papermaking and some specific procedures to pay attention to in order to maximize quality of the paper.

Overall, our re-shaping nature of the original materials corresponds with the process in papermaking that the shaping of pulp into sheets of papers. Mr. Gong also provided us novel viewpoints of choosing the appropriate material. We now can consider our characterization from different aspects. Mr. Gong also expanded our view on the aesthetic application of bacterial-cellulose. We can consider not only the practicality of our products but also the aesthetical nature and different designs of our creation.

Meanwhile, we have refreshed our understanding of environmental protection and sustainability that non-natural-degradable materials aren’t necessarily bad. Because the ultimate goal of modern technology is to create sustainable materials that use the least resources.

Dupont

In order to make up with our shortcomings in a lack of expertise in turning a research product into real life, we contacted with DuPont and learn some of the pros and cons of the traditional chemical synthesized material. According to this process, we not only had a general overview of the current material industry but also learned about the lifecycle of a product from design to mass production, which contribute to our experiment a lot.

RESULT AND CONCLUSION

We achieved the production of bacterial cellulose through static culture. There is visible bacterial cel-lulose in the culture flask with HS media. Apparently, the wet weight of bacterial cellulose produced by G. xylinus is much bigger than the dry weight according to the graph.

As for the production of bacterial cellulose by Rotary Disc Reactor, because of the current pandemic of COVID-19, we haven’t performed experiments in this part, but we are sure that the fermentation via RDR will be successful in the next iGEM competition.

We tried to purify SpyCatcher003 and SpyTag003 based on two chassis organisms: and P. Pastoris and E. coli, but the transformation and protein purification process for P. Pastoris are both trouble-some. Finally, we achieved the successful transformation of /P. Pastoris/ with genes responsible for SpyCatcher003 and SpyTag003 integrated with structural proteins according to genome PCR.

However, we didn’t finish the subsequent protein purification process due to the early closing of Bluepha lab. As for the protein purification from E. coli, according to the SDS-PAGE, the proteins are not successfully synthesized. Instead, they might be digested to become several fragments of poly-peptides with different length.

We choose to be a Two-Phase project for the next year iGEM competition. Our project next year would be based on the theoretical design and modeling prediction done in iGEM 2020, moving for-ward to experiment implementation if the pandemic were relieved.

METHODOLOGY

Production of Cellulose

We chose G. Xylinus to produce bacterial cellulose. And in two ways we tried to optimize the production rate: culture medium and culture device. We designed a dynamic bioreactor where G. xylinus can get both enough nutrients and sufficient oxygen to grow.
For medium, we first tested the Hestrin-Shramm medium, a traditional culture medium for G. xylinus. Unfortunately, it cannot provide G. Xylinus with enough nutrients for growth when G. xylinus is dynamically cultured in a large quantity1. Here is where the YME medium (Yamanaka Mathematically optimized Ethanol Media) is introduced; it can provide a lot more nutrients than HS.

Protein Synthesis in Pichia Pastoris

Figure 1. The plasmid expressing the CBMs with SpyCatcher003 in P. pastoris Figure 2. The plasmid expressing collagen-like proteins with SpyTag003 in P. pastoris Figure 3. The plasmid expressing spider fibroins with SpyTag003 in P. pastoris

Recombinant protein is needed to enhance the performance of cellulose. Three shuttle plasmids carrying the gene of our structural protein is first transfected into E. coli and colony PCR is used to select the successful colonies. Then after culturing, the plasmids are collected and transfected into P. pastoris, our chassis. When cultured with methanol as its carbon source, AOX1 promoter can significantly increase the production of recombinant proteins. Finally, we can purify our protein using nickel affinity chromatography and the 6*His tags that’s being synthesized along with the proteins.

TEAM ACHIEVEMENT

Hardware

Bacterial cellulose has been adopted in iGEM projects for many years as a polymer with a bright future in material science. However, methods for its production proposed by previous iGEM team are not ideal, that all those methods adopted static culture for the fermentation of G. xylinus, which is an extremely inefficient way for the biosynthesis of bacterial cellulose. Therefore, our method for the efficient production of bacterial cellulose would be a great contribution to the iGEM community.

Rotary disc reactor (RDR) has been proposed a long time ago. The core components of RDR are motor, an axis, and several plates. The axis is connected with the motor, while plates are anchored on the axis. Therefore, the rotation of the motor will drive the rotation of plates. It has been suggested in the literature that the adoption of RDR could significantly enhance the production of bacterial cellulose. Therefore, we decided to design and construct an RDR for the mass production of bacterial cellulose.

Accordingly, we have finished the assembly of the entire hardware, and the idling test proved that it could work well for more than 24 hours. See more details on our Hardware webpage.

Providing a platform for crosslinking of Bacterial Cellulose and various recombinant structural proteins

As a good piece of leather should be tensile, strong, and flexible. To achieve these standards, we cross-linked recombinant proteins critical to these facets between cellulose layers, the place where it is the weakest (Figure 3).

Figure 3 Interaction between recombinant proteins (created with BioRender.com)

Collagen is the main component of natural leather. So, we chose collagen-like proteins to imitate the properties of collagen. We also realized that leather should be wear-resistant and robust, and we synthesized spider fibroin to achieve this.

These functional proteins will be linked with elastin when synthesized. Proteins synthesized separately will be connected through suction filtration, trying to reach infinity affinity by SpyTag003 and SpyCatcher003 to speed the reaction up. To combine this network with cellulose, we use cellulose-binding modules (CBMs). As a fulcrum for the connection.

Through this platform made of variable recombinant proteins, Spytags and SpyCatchers, and CBMs, numerous projects related to BC, or even modified BC in areas of structural or colourific properties

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ACKNOWLEDGEMENTS, SPONSORSHIPS, AND REFERENCE

Acknowledgements

Ms. Hu
Mr. Gong

Sponsorships

We would like to thank to SnapGene and geneious for their licenses, New England BioLabs for their reagents and enzymes, and most important, revive & restore for their generous support and sponsorship. Additionally, IDT and Twist Bioscience provide each iGEM team in 2020 with 20kb and 10kb free gene fragment synthesis, respectively. They helped us to synthesize the DNA constructs wanted..