The principle of contributing to the iGEM and world community is key to our project. The nature of science is the expansion of the universal knowledge base through small increments made by individual contributors over a long timespan. This year, we played our role in helping to expand the iGEM knowledge base and contribute to future teams by contributing to the Parts Registry; identifying novel, replicable strategies for the optimized synthesis of defensins; "minesweeping" for infeasible design strategies based on our experimental evidence; and inspiring action from future teams to combat the pressing issues of war, wound infections, and antimicrobial resistance.

The iGEM Parts Registry is a perfect manifestation of the power of individual contributions of knowledge in academia, comprising the achievements of individual teams over the entire history of the competition. Every team could build on individual parts in the Registry, while in the process expanding the iGEM knowledge base by documenting new information about the parts they utilized and adding new entries to the Registry for future teams to utilize and improve upon. This year, we have added several new documentations to existing parts and created several new entries for a collection of basic and composite parts for the benefit of future teams.

New Documentation Added to Existing Parts

(Details of how we obtained the data within the added documentations could be found in our Engineering Success page)

We adopted the celluose binding domains CBM2 (BBa_K4011001) and CBM3 (BBa_K4011000), first documented by iGEM21_LINKS_China, as part of our collection of binding domains to allow the attachment of our antimicrobial fusion proteins to target first-aid material. Thus, we added our assessment of the binding ability of these domains by attaching them to chromoproteins and exploration of their potential application as a component of an antimicrobial coating to the documentations for these parts.

The α-defensin HD5 (BBa_K3924005) and β-defensin HBD3 (BBa_K3351002) were also first documented by iGEM21_Tsinghua and iGEM20_NWU-CHINA-A, respectively. As they were parts of the curated collection of defensins we focused on, we added documentation for the qualitative antimicrobial activity and quantitative minimally inhibitory concentrations of these defensins, measured by agar and broth dilution respectively. We also verified approaches to optimize the biosynthesis and purification of these defensins, which will be covered in detail in the "New Approaches" section.

New Basic Parts

We documented all previously undocumented binding domains that we adopted, namely the collagen binding domains α1 (BBa_K5185003) and α2 (BBa_K5185004); chitosan binding domain CBM5 (BBa_K5185002); and alginate binding domain CBMxx (BBa_K5185008). As part of our proof of concept design, we determined the binding activity of these domains, which could be helpful for future teams developing fusion proteins that bind these materials for other purposes.

We also documented all previous undocumented defensins that we adopted, namely the α-defensins HNP1 (BBa_K5185000) and HNP4(BBa_K5185001). We included measurements of their antimicrobial activity obtained from agar and broth dilution, which could be helpful for future teams adopting these defensins as part of their design.

Moreover, we documented improvements to part HNP1, namely the optimized HNP1Ala (BBa_K5185028), HNP1AWW (BBa_K5185030), and HNP1AWK (BBa_K5185031). These improved parts were optimized through individual amino acid mutagenesis on the basis of increasing their dimerization likelihood, cationicity, and hydrophobicity to enhance their antimicrobial activity and stability. However, our results showed that the optimized defensins displayed lower antimicrobial activity than their original counterpart. Thus, this was part of the "minesweeping" we conducted, warning future teams that plan on optimizing defensins that an optimization approach focusing solely on the factors we selected was likely too simplified and infeasible. More "minesweeping" based on our failures is described in detail in the "Minesweeping" section.

New Part Collection

The basic parts that we documented or added documentation to collectively formed an arsenal of binding domains and defensins that could be assembled into a collection of composite antimicrobial fusion proteins when the SUMO proteolytic cleavage tag is added in the middle of the two domains. These fusion proteins would be capable of forming antimicrobial coatings on a wide range of first-aid materials and imparting antimicrobial activity when the Ulp1 protease is added to cleave the SUMO tag. As defensins possess bioactivity against additional candidates such as viruses and cancer cells that we did not focus on, the collection of fusion proteins could provide a well-rounded toolkit for future teams that wish to utilize defensin binding for unique applications. Our mode of fusion protein synthesis also prevented the issue of host toxicity and proteolytic degradation faced when defensins are synthesized by themselves. This new approach could act as inspiration for future teams that wish to biosynthesize ultra-short peptides that would cause adverse effects on the host when synthesized independently.

Throughout our design-build-test-learn (DBTL) cycles documented in our design page and outlook page, we unfortunately experienced multiple failures. However, failure could also act as a strong impetus for change. We analyzed and summarized the causes of instances of failure in our design to act as a "minesweeping" reference for future teams whose project might adopt components similar to our design.

Aside from the sequence optimization attempts of HNP1, we also identified that out of the tested binding domains, CBM3 and CBM5 possessed relatively high solubility, and α2 possessed relatively low solubility. We came up with this conclusion because the fusion proteins containing α2 were extremely hard to purify compared to those containing CBM3 and CBM5. Thus, future teams aiming to synthesize fusion proteins containing α2 would likely have to implement measures to increase the protein's solubility in order to achieve favorable yields.

Lastly, from experience, we determined that the optimal fermentation conditions for our fusion proteins is to induce the SHuffle T7 E. coli cells when the OD50 value of the media is 0.8-1.0 by adding 0.1 μM of IPTG and allowing the culture to ferment for 10-12 hours at 250RPM, 20°C. When we experimented with other values for the parameters, the fusion proteins are often found in unfavorable inclusion bodies. This suggested that the amount of dissolved oxygen within the culture medium, which the mentioned parameters affect, is likely correlated with the tendency to synthesize the fusion proteins into inclusion bodies. Thus, future teams wishing to ferment fusion proteins containing defensins could draw on the fermentation conditions we determined as reference to ensure optimal yields.

More importantly, beyond the world of iGEM, our project is built on the need of action in light of the pressing global crises of war, wound infections, and antimicrobial resistance. Our description of the severity of these alarming issues could spark action in future teams around the world to tackle these problems in new, creative ways that best benefit their local community. A spark can turn into a flame, a flame a fire. Local solutions to local problems could together form an interwoven web of protection on a global level to protect humanity against the concerns of war, infections, and antimicrobial resistance.