In our experiment, we initially chose two different chassis to express defensin: Pichia pastoris yeast and E. coli. Ultimately, we selected E. coli to be the chassis. This was not because Pichia pastoris couldn’t express defensin, but because the yeast experiment cycle was too long due to its complex culture conditions and fermentation process. In addition, the relative growth rate of Pichia pastoris also takes more time compared to E. coli. By the time we completed constructing the yeast strains, the fusion protein expressed in E. coli had already been successfully verified for its function.

However, the yield of the purified fusion protein in E. coli was unstable, as a portion of the target protein was trapped in inclusion bodies. Even though we optimized factors such as fermentation time, temperature, and IPTG concentration, we were still unable to eliminate the formation of inclusion bodies. Furthermore, we also attached two soluble tags to the N-terminus of the target protein to improve solubility, but the results were not desirable since there was no significant increase in the production of purified protein (Fig. 1).

Given these challenges, we believe that Pichia pastoris will be a promising alternative to E. Coli. This yeast system offers several advantages over E. coli, particularly in terms of protein secretion. One of its main strengths is the higher yield of secreted protein. Pichia pastoris can secrete proteins directly into the culture medium, which significantly simplifies the purification process and can lead to a higher yield of defensin (Zhang X，2018).

In contrast, E. coli often needs complex purification procedures, in which the expressed proteins tend to remain inside the cell or form inclusion bodies. This characteristic of Pichia pastoris not only simplifies the purification process but also reduces the possibility of inclusion body formation, potentially addressing the main issues we encountered with E. coli.

Overall, These factors of Pichia pastoris makes it an prospective option for our future work on defensin expression.

In high-risk environments such as battlefields, medical supplies must be adaptable to diverse conditions. The inclusion of multiple domains and healing material would ensure to address different medical needs, providing support for wound healing and infection prevention. Beyond the range of defensins, a wide variety of antimicrobial peptides can also be incorporated into the functional domain of the fusion protein. This includes peptides with specialized functions such as those targeting tissue regeneration or inflammation, which could lead to multifunctional wound dressing capable of not only fighting infection but also giving more functions such as improving tissue reparation and minimizing scarring.

While our current focus is on first aid kits for emergency and battlefield use, the potential applications for these materials are immense. For instance, the antimicrobial properties of our product can be added to daily items such as food packaging to extend the shelf life and prevent food from bacterial contamination. Not only in food packaging, but it is also effective in many other products like clothing, medical supplies, and some skin care products.

In a world with active small conflicts and natural disasters, our project offers a sustainable and innovative solution to fight against antibacterial resistance. Furthermore, the potential impact of our project extends beyond the battle and medical field with the integration of antibacterial peptides into everyday items, ultimately benefiting communities and improving public health on a global scale.

Our domains can also be used for other purposes besides antimicrobial connectivity. CBMs are specifically designed to recognize and attach to cellulose, which is a key component in cotton and other natural fibers. By fusing CBMs with chromoproteins, it enhanced binding to cellulosic fibers. The resulting chromogenic fusion proteins also exhibit stronger strength of binding to these textiles, ensuring long lasting colors. This allows a more sustainable dyeing process by reliancing less on synthetic dyes and harmful chemicals. Since chromoproteins are derived from biological sources, they give an eco friendly alternative to traditional textile dyes, aligning with the belief of specific group of people and sustainability in the textile industry. Additionally, the binding properties of CBMs allow for reversible dyeing, meaning colors can be removed through washing or other treatments. This feature provides flexibility for consumers and reduces textile waste.

Moreover, the incorporation of CBMs enhances color maintenance, with studies showing that the use of polyethylene glycol in conjunction with CBM fused chromoproteins significantly improves the longevity of color on fabrics, addressing a common issue in textile dyeing. This Year, we did some fun experiments. We made fibers capable of being used for weaving by wet spinning CBM3-sfGDP with cellulose, a method that requires a 2-hour immersion in 60°C waters, and we were concerned that this method of making fibers might lead to degradation of protein. However, we were surprised to find that CBM3-sfGFP could make the fibers fluoresce green!

Alginic acid, derived from brown algae and certain bacteria, is a highly beneficial biopolymer for wound healing due to its unique properties and versatility. Its hydrophilicity allows it to absorb large amounts of liquid, making it effective at managing wound secreta and creating a moist environment that promotes tissue formation and healing while reducing the risk of infection. Alginate does not strongly adhere to cells or skin tissue. Due to the special structure formed by alginate and calcium ions, combine with the carbon dioxide in carbonated water that prevents acidification, the resulting novel hydrogel not only exhibits ideal pH and humidity conditions favorable for wound healing but also has significantly lower adhesion and swelling rates compared to other hydrogel dressings that have achieved commercial application.

Furthermore, alginic acid helps control bleeding during the early stages of wound healing. It is non-toxic and does not provoke an immune response, making it a safe option for patients with sensitive skin or allergies. In addition, alginic acid is environmentally friendly, being seaweed-derived, and offers an alternative to animal-derived materials like collagen and chitosan. The material's biodegradability enables it to be naturally broken down over time, which makes it more beneficial for the sustainability of the environment. This property also helps gradually release medicine such as antibiotics or growth factors directly to the wound site, reducing the need for surgery to remove the dressing. All the features of alginic acid makes it an prospective healing material.

In our experiment, We test multiple kinds of combining domains of aligning acid including CBM13, CBM16, and CBM32. However, due to contamination issues in the later stages of the experiment, we only chose to test the newly discovered CBMxx.

With the increasing development and promise of the AI field, It is possible that AI can be used to find the common features of these material binding domains and develop binding domains that can bind multiple materials.