Entrepreneurship

At least 46% of the Great Garbage Patch is nets, which are made from nylon and do not biodegrade. Nylon nets are abandoned when they break suddenly or get tangled, and harm wildlife by tangling around them, destroying their habitats and releasing microplastics. Fishermen who use nylon nets have no choice but to discard their nets when they get tangled or broken, as there is no way to fix them. We propose nets that indicate when they are close to breaking, can be fixed, and are biodegradable if they do end up in the ocean.
Our Caddisilk project has three integral components: spider silk, silkworm silk, and caddisfly silk. Previous research has successfully expressed spider and silkworm silk proteins in yeast and bug cell lines. Notably, spider silk offers a highly viable production path, with artificial, modified silk available that is much more feasible for biosynthesis compared with spider extraction. Additionally, silkworm silk has a long history of usage in textiles, making it easy to acquire and allowing us to reduce the net cost of our nets.
This leaves the team with the task of bio-synthesizing caddisfly silk. With the support of experts in caddisfly silk proteins, we have designed, constructed, and successfully transformed our caddisilk plasmid into E. coli. As we shift our focus to scaling up production and protein purification post-expression, our work has prepared us for the upcoming phases of the project.

Regulatory organizations have tried to reduce the amount of microplastics being thrown into the ocean. In additon, environmental protection laws have been swaying the general public to care more about sustainability, and this should ultamitely impact fisheries as well. There has been a shift culturally to do more about the growing problem of microplastics in our ocean, and protect our environment not solely thorugh an individual basis, but also through larger companies who have the greater emissions and harmful impact on the environment. Our startup, SilkSnare, seeks to capture this shift in market by providing a biodegradable and stronger solution for fishing nets.

Biodegradable fishing nets are not a viable option for commerical fishers in the status quo. Recent field trials conducted in Norway and South Korea show that biodegradable nets catch significantly fewer fish than synthetic ones, which scientists hypothesized was due to the material's greater elasticity. The longer the biodegradable nets were in the water, the more they broke down and the less efficient they were at catching fish. Fishing nets need to withstand harsh environmental conditions, resist abrasion, and endure repeated use - biodegradable materials available on the market are simply not up to this challenge. Furthermore, some biodegradable materials don't perform as well as synthetic materials when wet. Factors like water absorption, buoyancy, and degradation in aquatic environments can impact the reliability of biodegradable nets in fishing operations. Caddisilk provides a solution to these hurdles, enabling environmentally friendly fishing to enter mainstream commercial practice. Our base net will be interwoven spider and silkworm silk for their characteristic strength and toughness. The true innovation lies in the coating of caddisfly silk that provides water resistance, long lasting durability, and stiffens the fibers to decrease elasticity. Since caddisfly silk is naturally suited to acquatic environments, it is the key material in creating the fishing nets of the future.

SilkSnare not only boasts biodegradability and reduced cost, but durability paralleling current synthetic nylon nets; this same durability that popularized synthetic nets in the first place. Previous studies have cited that the true cost of implementing biodegradable nets being economic. With a reduced pricetag, SilkSnare would increase profits among fishermen by reducing material costs while maintaining net efficacy. To push incentive further, the growing megatrend of sustainability (think WholeFoods, electric vehicles, reusable bags) would allow fishermen to up mark the price of their fish—in similar fashion as organic groceries—which would now have a sustainable pricetag. Thus, SilkSnare proves a significant financial incentive for all fishermen. With an estimated 4.1 million fishing vessels in 2020, fishermen are SilkSnare’s largest market opportunity. Government agencies such as the EU Commission and the US EPS may also have interest in investing in SilkSnare’s environmentally-conscious goals.

In the fishing net industry, stakeholders encompass a diverse range of groups, including fishermen, fishing industry associations, environmental organizations, government agencies, suppliers, retailers, distributors, and consumers. Fishermen, as primary users, directly influence product design and functionality. Fishing industry associations advocate for industry standards, shaping market practices. Environmental organizations and government agencies drive demand for sustainable fishing net solutions, giving us a reason to produce these nets in the first place. However, they may also place restrictions on the nets, so we will need to work to meet their constraints. Suppliers and retailers will influence production capacity and quality, so we will need to compete against them to validate how our solution is more viable. Finally, consumers' preferences and purchasing behaviors drive demand and innovation. These stakeholders collectively shape the initial target market by influencing product offerings, market practices, regulatory frameworks, and consumer perceptions.

Our first verification of the problem was through going to the IGEM competition (Jamboree 2023), where we were able to talk to dozens of industry experts as well as judges to verify the need for the development of caddisilk, as well as its most needed applications. Since the material has a lot of unique properties: such as being water-repellent and durable, we explored a variety of options for its application in various industries, and after getting professional opinions we settled on the sustainability applications of CaddiSilk: resolving the problem of environmental waste while accommodating commercial and recreational needs in order to keep the fishing industry going but reducing the ocean pollution through development of a material that is biodegradable and non-cytotoxic. We were also approached by several professionals (for example, from the APL), who approved the idea and confirmed that there is indeed a need for caddisilk products on the market. Overall, we were able to gather that there is a great interest in the industry from employers and other businesses, as well as industry professionals. Throughout this process, we have also been receiving mentoring from the faculty who have been able to evaluate the idea and have also confirmed the need and potential profitability of this project.

In order to address the issue, we are developing the Caddislik material from readily available E. coli bacteria. The process of bio-synthesizing caddisfly silk, includes modification of the E. coli strain to develop the relevant protein that would allow for the bacteria to imitate properties of the naturally produced Caddisilk material. Through the development of the technology to produce this material in vitro, we will be providing industries with a scalable process, that is also a sustainable practice and will allow the markets to attract more customers due to the recent growing trend in environmental awareness. The product is both the technology and the material that will be used for the creation of fishing nets. It is extremely hard for competitors to copy our solution because of the unique mutation that leads to a specific protein, which has been in the works for over a year now. Caddisfly are also not readily available for testing and are not well known. While we were doing our initial literature search, we discovered that there is an extreme lack of research done into these organisms, which provides us with a competitive advantage and a label of a new breaking technology.

This net design can be used to make both large trolling nets for commercial fishing ships, and smaller nets for small-scale fisheries and recreational fisherman. Once purchased, the only regular upkeeping our net would need is redipping or potentially spraying on caddisfly silk, in order to maintain the net’s waterproof coating.

Historically, commercially used plastic netting has led to 81,130,112 lbs (36799999 kg) of waste in addition to causing a decline in fish population from discarded fishing equipment. Plastic netting also releases chemical waste when it is both produced and left in the ocean. SlikSnare provides an environmentally friendly, durable, and heat/water-resistant silk net alternative. While SilkSnare is not the first silk net that was made to solve the problems of plastic netting, it proves itself to be the most effective compared to its competitors. Rather than using spider or silkworm silk compared to other biodegradable net companies, SilkSnare uses caddisfly silk instead, which has better durability and strength. This fixes the problem that many biodegradable companies face, which is their lack of efficiency compared to plastic. Additionally, SilkSnare is reusable compared to the other net alternatives as the cost of fixing the net is cheaper than buying a new net.

We do have some proprietary advantages, in that our protein design of the caddisfly silk is unique; in fact we would be the first to express caddislfy silk in E. coli. Other key competitive advantages are that we would be able to easily evaluate the strength of the remaining strands of the net, redipping our net to restrengthen it would be at a lower cost than buying a new net, and the fact that our net is biodegradable means our product would allow local fishing populations to rebound, enabling fishermen who use our product to become more profitable over a longer period of time.

We would like to begin our journey with a local fishery in the Chesapeake Bay area, since that will give us the most direct way to impact our local community since they are also invested in fishing sustainably and providing minimal risk to the local ecosystem

Our plan for validation of solution will be to take advantage of our close proximity to the Chesapeake Bay, and temporarily loan out a net for free to local fishermen and fisheries, so as to allow local stakeholders to test the advantages of our product for themselves. We will use feedback from those in the Chesapeake Bay community to help further improve our product wherever needed. A second facet of our validation plan will be using customer discovery through I-Corps which is funded by the NSF, to conduct user interviews with 100 people and determine if we are actually addressing the problem in a way that is helpful and impactful.

The best way for our product to acquire customers is to focus on our target audience and gain their appeal. The product that we are offering is a fishing net made entirely of biodegradable silk. For this product our customer segment that we will be targeting is the fishing industry, the United States Fish and Wildlife Service, and the National Oceanic and Atmospheric Administration (regarding the latter two, government scientists will be interested in a new environmentally friendly way to design trap nets for aquaitic surveys). In order to get our product out into the market we would first do this by having multiple market locations. Our plan is to first reach out to local fisheries in the Chesapeake Bay area which is lcoal to us, and receive feedback on our prototype solutions and ways to improve the design. They will also serve as our first customers, and hopefully our solution will gain traction as a better biodegradable solution compared to what already exists. After that, we will continue to reach out to fisheries and work our way up to larger scale companies, to deploy our solution once we have a proven model for sucesss.

"Purchasing silkworm silk will cost around $15 per kilogram (1), while the cost of synthesizing spider silk from E. coli would cost $23 per kilogram (2). The cost of synthesizing caddisfly silk from E. coli would be slightly more expensive than synethisizing spider silk, likely around $30 per kiilogram, simply due to more nucleotide sequences needing to be ordered for genetically modifying the bacteria to express caddisfly silk proteins, compared to spider silk protein. Since silk is 20% less heavy than plastic lines (3), a 300 ft commercial fishing plastic net weighing at 13.6 kg (4) would only be 10.9 kg as a silk net of the same size. In order to produce 10.9 kg of silk - 1/3 silkworm, 1/3 spider, and 1/3 caddislfy - production costs would end up being minimum of $247.07. Our price, therefore, would be $350, giving us a little over $100 of revenue above operational costs per net.

"We have discussed our pricing plan with Josep, our HopStart mentor, and this plan generally makes sense considering we are trying to emphasize how redipping the net in the solution will help our net rest at an equivalent if not cheaper cost than the current cost of a non-sustainable net. Additionally, our team members have competed in GreenHacks, the sustainable hackathon at Johns Hopkins, to win honorable mention with $400. At the event, we discussed our strategy with industry professionals and judges who also helped validate that this pricing solution would make sense long-term."

We plan to address a large portion of the fishing market industry, particularly local fisheries. If this is successful, we hope to expand to more prominent and larger-scale fisheries, as we scale our operation to mass produce the silk. Another market we see large promise for is the medical industry with silk sutures or endogenous wound healing, since our proprietary caddisfly silk is also biocompatible, and we believe hospitals would be willing to pay a premium for a material that shows promise in serivng as a safer alternative to existing materials. However, this will require more rigorous testing, patenting our design, and additional approval from the FDA, which will take, money and resources. Thus, we believe that after proving our silk in the fishing industry, we would have greater means and experience to puruse the medical industry, where our silk could change lives. We see caddisfly silk growing into a brand that provides solutions for different industries."

We have read previous research articles where spider silk can be used as a biodegradable thread in surgery for sutures. From speaking with Dr. Michael Kessler from the Materials Science department, it is clear that hospitals would be willing to pay a premium for thread if it meant it could be safer/more effective for the patient. We would need to conduct further studies, but we would be able to reach stakeholders in this market relatively easily considering the Hopkins medical network. We have some validation that the medical market can be reached and addressed, primarily from a design challenge by Johnson and Johnson to innonvate a solution for post-operative surgical infection management. As we develop our startup, we can validate this further with professionals from the Johns Hopkins School of Medicine, who may be able to give us greater insight into how we can address the surgical infection space through caddisfly silk sutures and wound sealants."

Our one-year development plan is as follows:
May 2024 - express caddisfly silk protein through wetlab experimentation
June 2024 - validate and characterize using adhesion and heat tests, and biochemical assays
July 2024 - order silkworm silk and express spider silk thorugh E. Coli
August-September 2024 scale-up using a bioreactor
October 2024 - combine three silks using fishing net braiding machine
November 2024-January 2025 - test working prototype for tensile strength, optimize percentage of silk combinations, thickness of net, continue braiding production
February 2025 - reach out to stakeholders with prototype, refine design with feedback and send out samples to Chesapeake Bay fisheries
March 2025 and onwards - continue with development plan as specified earlier
Some CSFs for us now include:
1. Expressing caddsfily silk sucessfully using E. Coli - otherwise, our idea to braid and optimize all three silks will not succeed. This comes with a moderate risk, as if any of our core wetlab members leave, we will have a much harder time finding the time and resources to produce this silk. However, we also have a contingency plan in training new members in the wetlab, so we can always have people working on developing caddifly silk.
2. Acquiring the capital to purchase the bioreactor for producing enough silks, purchase the nickel-beads and gravity columns for protein purification, and purchasing the fishing net braiding machine to weave the purified silks together to make a net.
3. Paying for patents would be another key success factor.

From a marketing stand point, we are assuming that local fisheries will be receptive to this design and willing to pay a slight initial premium for a sustainable alternative to current fishing nets. We will test this however, through our participation in the I-Corps program as described above, to ensure that we are addressing the needs of our customers. For operational costs, we are assuming that the cost of materials and especially silk, does not change drastically from now until it is time for us to produce. We have factored in however, the cost to perform tests on our nets to ensure tensile strength and viability in the water for fishing.

Ideally, we will be filing for a provisional patent to protect our product in its early stages. Until we receive the full patent, we will not be able to sue for any infringement, however, we are confident we have a strong idea to be patented, so once we receive the patent, we will be able to fight it retroactively. We also plan to partner with local fisheries in the area and demonstrate how our fishing net is far more environmentally sustainable, giving us an edge over our competitors. Larger corporations will immediately have a difficult time replicating our net, as if we are successful with caddsifly silk production, we will be the only people in the world who can produce the caddsifly silk, which is proprietary to our design, giving it the elasticity and biodegradability unique to our net.

In the fishing net industry, there are not many regulations that we would need to comply with aside from some minro fishing net size requirements. This should not be an issue for us, as we can design our nets to fit these requirements, and additionally scale them to meet the needs of any size the fishery desires.

We currently do not have any explicit intellectual property created as of now, but any intellectual property or patent that would be created would be owned by the members of the team. This is because we designed the gene sequences for the synthetic Caddisfly silk, as well as originated the design for the net as a whole, and did not rely on direct outside assistance or contribution, so we would be able to keep the intellectual property contained to our team.

The operational costs would be $3500 to acquire a fishing net braiding machine, in order to braid our silk strands into a net once we have produced purified silks. Costs for advertising will be minimal, since initial rollout will involve personally reaching out to local Chespeake Bay stakeholders.

We would like to acquire a patent on our technology, so we estimate around $10,000 to start the patent application process in the long-term. Aside from this and associated legal fees, we do not anticipate any other major product and marketing costs with our design.

We would invest in software resources for more powerful modeling that can support our protein size, reagents and bioreactor equipment for the scale up process, and professional plasmid synthesis for alternative protein designs.
First, we need at minimum, an additional $1500 to purchase materials essential for completing assembly and protein purification stages of our synthetic Caddisfly silk design. This funding is crucial for advancing to the characterization phase, where we will conduct adhesion and heat tests on the derived protein. Understanding the scalability of protein production is also a priority; hence, extensive biochemical assays will be necessary. These assays will evaluate critical factors like the optimal pH for protein folding, appropriate polymerization temperatures, and suitable buffers.
For protien purification, it would cost us around $500 for nickel-beads and gravity columns to do His-Tag protein purification; as well as another $500 to build a silk glue gun, which will add sheer force to convert the purified silk protein solution into solid silk strands.

We are currently relying on small university grants ranging from $500 to $1500. These funds come from departments at Johns Hopkins including the Biomedical Engineering and Materials Science Deparments, the Alumni Association, and Student Government Association. We are also applying to business plan competitions and hackathons, and have already received $400 from GreenHacks in support of this sustainable alternative.
Long term, we will need a greater amount of capital to cover our operational and production costs as described above. Once we have a stronger demonstration of the silk net itself, we will look into more sustainable forms of funding, such as a line of credit. During our time in developing this idea, we will continue to look and seek opportunities with investors, to ensure that we are backed by people we trust.

Since we are still in the experimental stage of our product, we anticipate it will take another 6-8 months to develop a working prototype and then an additional 2-3 months to fully validate and test our product. Then, a year from now, we will be deploying our prototype to local fisheries. We anticipate it will be at least 1.5 years before we generate any revenue assuming that our initial deployment is sucessful, and about 2-3 years before we are cash flow positive.

Siyona - head supervisor: leading and managing the project timeline, meeting with project advisors and stakeholders
Vara - assistant supervisor and treasurer: leading and managing the subteams, assisting with logistical tasks, emailing, managing our team budget
Lucy - drylab co-lead: leading the computational and modelling portion of the project, has experience in creating a startup, managing teams, and holding team members accountable
Nicholas - wetlab co-lead: leading the experimental portion of the project, planning meetings, literature review, documentation in Benchling
Alex - wetlab co-lead: leading the experimental portion of the project, literature review, assists with experiments, researching theoretical solutions

From the Department of Biomedical Engineering, Dr. Winston Timp and Dr. Jessica Dunleavey, serve as our advisors and provide us with basic wetlab equipment and resources.
Additionally, last spring, we arranged a call with Professor Russell Stewart from the University of Utah, one of the world's leading experts in caddisfilies. We decided on project goals, discussed caddisfly silk and gene properties, and predicted major obstacles. He also shared an unpublished article with information on the modular structure of the h-fibroin gene, which is critical to the success of the team.

Caddisflies produce a very unique silk that maintains adhesive properties even when submerged underground. Additionally, it is resistant to heat and is non- toxic to human cells. Existing synthetic underwater adhesives contain chemicals like PFAs that are toxic to the environment. Our CaddiSilk would be a sustainable biomaterial alternative as it would be produced and scaled using synthetic biology without the use of silk worms or unethical human labor. CaddiSilk has many applications, ranging from thread that can be used in sutures to surgical adhesives.

Of the 64 million surgeries that are conducted in the United States each year, an increasingly large proportion of surgeons are choosing to seal wounds with surgical adhesives over traditional sutures. Sutures have a number of shortcomings, including: 1. Time-consuming 2. Traumatic punctures to tissue, making it a difficult choice for soft tissue 3. Positioning, which is difficult for surgeries conducted in smaller spaces (ie. laparoscopic surgery). Caddisilk seeks to bypass these shortcomings with a surgical adhesive that is non-toxic, waterproof, heat-resistant, and durable.

Caddisflies are insects whose larvae produce a silk protein fibroin - a heterodimer consisting of a heavy chain and a light chain. This silk forms networks of fibers that adhere to small objects and are resistant to high temperatures – up to 242 °C, resilient to deformation, and not cytotoxic to human cells. Most importantly, the silk is water-resistant and highly durable, making it a viable candidate for internal wound sealing. Wound sealants need to be leakage resistant, durable, easily applied, quickly curable, and sterile. CaddiSilk fulfills these requirements with its adhesive strength underwater, curing within 12 hours, and can be applied laparoscopically through tubes. It also degrades slower than current hydrogels and is non-toxic for humans. We plan to produce caddisfly silk in mass quantities by expressing the silk protein in E. coli.

Synthetic production of spider silk and silkworm silk have already been proven to be viable options, indicating high likelihood of success for expressing Caddisfly silk. For the past year, with the support of experts in caddisfly silk proteins including Professor Russell Stewart from the University of Utah, we have designed, constructed, and successfully transformed our CaddiSilk plasmid into E. coli as the first phase to test our solution. We are also working on protein models and visualizations to assess the viability of our solution at each phase, as we make modifications and resolve challenges in the wetlab process. As we shift our focus to scaling up production and protein purification post-expression, our work has prepared us for the upcoming phases of the project.
In the past year, we have also competed internationally at the International Grand Jamboree, hosted by iGEM, where we received a gold medal distinction for our work among 400+ other synthetic biology teams and received positive feedback from the judges. For funding, we have received numerous grants (see question 16) to support our work. Currently, we are discussing plans for collaboration with the Johns Hopkins Applied Physics Laboratory (APL) on the project, and we are also participating in the HopStart program and to refine our business plan and market strategy in anticipation for the pitch competition.

To succeed, our project requires specific resources which require greater long-term capital. We are currently relying on small university grants, but these take a significant amount of time and energy among our team leads to acquire, and ultimately decreases the time our team can spend on working on the experimentation and validation portion of CaddiSilk to advance the product.
First, we need at minimum, an additional $1,500 to purchase materials essential for completing assembly and protein purification stages of our synthetic silk design. This funding is crucial for advancing to the characterization phase, where we will conduct adhesion and heat tests on the derived protein. Understanding the scalability of protein production is also a priority; hence, extensive biochemical assays will be necessary. These assays will evaluate critical factors like the optimal pH for protein folding, appropriate polymerization temperatures, and suitable buffers.
We are facing several other significant challenges. One of the main technical obstacles involves the phosphorylation of serine amino acids to induce a negative charge on the protein, enhancing its stickiness. To address this, we are considering alternative strategies such as replacing serine with aspartic acid or other synthetic amino acids, or utilizing a kinase enzyme to achieve the desired modification. Moreover, our project is hindered by insufficient computational power, which is vital for the modeling of protein structures and the in silico assembly of subunits. This lack of computational resources severely limits our ability to carry out complex simulations and modelling required to validate our project viability. With this funding, we would be able to purchase greater reagents/lab materials for experimentation and stronger modelling software, which would take us many steps closer to successfully producing CaddiSilk.

We will begin marketing our product to research hospitals and medical schools who invest in innovation and research, so that they can investigate the applications of caddisfly silk in medicine. Ideally, this will establish caddisfly silk as a reliable biomaterial and allow us to widen our market to include all institutions that practice this type of surgery. While we anticipate CaddiSilk will come at a premium in comparison to other silk sutures on the market, we believe because of its greater biocompatibility and heat-resistant properties, hospitals would be willing to pay more to purchase these silks.
Our current revenue model is B2B, in which we license our product first to local hospitals in the Baltimore area for preliminary testing, and then expand to the general hospital market when we are certain of its efficacy. Outside the medical space, we see large implications for our product in the fishing space, providing ESG solutions in a space occupied by inhumane and unsafe fishing practices.
Long-term, we hope to file for a patent and gain FDA approval, which we understand is a long term process and will take more capital initially. However, we hope to eventually exit as we license the technology to a larger biotech company, since we envision CaddiSilk will be integrated within other medical products beyond surgical sutures and hope it can reach as many patients as possible.

We would invest in software resources for more powerful modeling that can support our protein size, reagents and bioreactor equipment for the scale up process, and professional plasmid synthesis for alternative protein designs. See question about challenges for more details.

We hope to finish the wetlab experiment portion and/or make more progress to validate that we can successfully produce this caddisfly silk synthetically. We view I&E as a fantastic opportunity to grow our entrepreneurship and startup knowledge and intuition, as we are a team of multidisciplinary scientists first, and have learned the business plan skills along the way. Mentorship and community from other I&E participants would be critical to our success as we navigate the space.