In addition to research, Yale iGEM explored diverse questions beyond the bench, including industry applications of AMPs in shipping and the interface between research and entrepreneurship. We also engaged high school students in a classroom discussion on the history and importance of AMPs.

Over the summer, Yale iGEM member Cathy Ren interviewed Mr. James S.C. Tai, Technical Director and General Manager of Fleet Management Department, Orion Overseas Container Line Limited (OOCL). We asked about the importance of biofouling in the shipping industry and the possible application of our antimicrobial coating.

CR: What is biofouling and why does it matter to OOCL?

JT: Marine paint has always been focused on protecting against water damage, which is called anti-corrosive, but more recently has moved more towards anti-fouling.

Biofouling can be caused by both micro- and macro-organisms. Microorganisms like bacterial slime can adhere to ships' hulls, while the macro-organisms include barnacles and algae. Barnacles in particular can be extremely corrosive. They release a type of acid that will eat through the paint and even the metal, embedding themselves deep in the hulls so that even scraping them off may not be effective. Usually, after scraping, another layer of paint will be applied, but the paint cannot adhere well to a hull that still has these embedded barnacles attached, as the organisms chemically react with the paint.

"...the organisms that attach to the hull will increase the weight of the ship, sometimes enough to double fuel costs..."

Biofouling is an important problem because the organisms that attach to the hull will increase the weight of the ship, sometimes enough to double fuel costs if the ship remains in warm waters for an extended period of time and organisms really begin to build up. In just a few days, barnacles can attach and build up enough to pose an enormous challenge to the removal process.

CR: What are the anti-fouling methods that OOCL employs?

JT: There are two main anti-fouling methods currently in use. One is application of a particularly slippery paint, to make the surface mirror-like, so that organisms can't attach to the hull in the first place. However, this paint is very expensive, and application must be done in very specific conditions, like a specific temperature of water, and so on. However, if ships remain immobile, the slipperiness of the paint is not enough to prevent biofouling. And it would be nearly impossible for us to run a shipping company if the ships never docked for unloading. Ships will have to stop eventually, so this method is not completely effective. Also, any damages to the surface, for example from bumping into something while docking the ship, will render the slippery properties useless.
Furthermore, this mirror-like surface, while it can prevent barnacles from attaching, cannot escape things like slime.

Another anti-fouling mechanism is through the use of biocides, which kill organisms like barnacles, but these will also kill other marine organisms so they are not environmentally-friendly. Tin was once the standard biocide, but the industry has since moved to copper, which is supposed to be less harmful to the environment somehow.

These biocides are incorporated into a type of paint that will fall off the ship when the ship moves at a certain speed, so any barnacles that have somehow adhered will fall off. One problem is that even if the organisms are killed by the biocides, a process that requires a certain amount of time, their shells may still stick to the ship so the paint- falling-off process is necessary. Then this paint will fall and remain in the ocean, killing other organisms.

CR: What are some other methods for preventing biofouling that are currently being explored?

JT: Some people have proposed regular polishing of the hull to work quickly and prevent heavy buildup of barnacles, but this is both time-consuming and expensive. Although anti-fouling measures are necessary, we can't forget that shipping is the main service, and too much cleaning may not be profitable.

A strategy not yet on the market, still undergoing research, is using a paint that signals to the barnacles that it is attaching to something not stable, like water, so that it will not try to adhere to the hull. However, it is uncertain how this would apply to things like slime and algae.

CR: Yale iGEM is currently trying to produce an anti-microbial protein that could be used in the field of anti-fouling. What are some things we should consider with our approach?

JT: iGEM's protein product is exploring an area of opportunity and innovation. You will need to consider how the protein will affect different types of organisms -- algae, slime, barnacles -- and whether it will work in various temperatures. You will have to consider how the coating will be created and applied to the ship, like will you incorporate it into the paint? Also, even though the main purpose of the protein is anti-fouling, it must not interfere with the anti-corrosive paint, which is considered to be more important than biofouling.

The anti-microbial properties of the protein will need to work before the barnacles release acid and attach to the ship because even if barnacles die, their shells will remain.

Hopefully, the protein will be biodegradable, as that would make it more likely to be accepted by environmental authorities.

The paints and coatings applied to ships are very specific. Different coatings are used for ships that travel in different waters with different organisms, so any protein product used for the treatment and prevention of biofouling would optimally be able to work in a wide range of temperatures and waters.

"any protein product used for the treatment and prevention of biofouling would optimally be able to work in a wide range of temperatures and waters.

You could also look at bridges: barnacles also attach to bridges, and their acid corrodes the metal, making the bridges less stable. Bridges are also immobile, so you can't use the slippery-paint method.

(Disclaimer: The interview was translated from Mandarin, so some terms/translations may not be completely correct.)

Yale iGEM Interviewed Dr. Christopher Loose, co-founder of Semprus Biosciences, on entrepreneurship and using research to address unmet needs in medicine.

Dr. Loose co-founded Semprus BioSciences, a company focused on anti-fouling on medical devices, with MIT professor Robert Langer and David Lucchino in 2006. He served as CTO until the company was acquired by Teleflex Incorporated in 2012. He currently serves as Executive Director of the Yale Center for Biomedical and Interventional Technology (CBIT) and as Assistant Professor Adjunct, Urology and Lecturer, Biomedical Engineering and Lecturer School of Managment.

iGEM: As someone with extensive experience using anti-microbial peptides to target industry needs, what are some challenges with bringing an AMP-based solution to market?

CL: First, a lot of work has been done in this area. This means there are many papers and patents on attaching AmPs to surfaces. Two additional challenges are costs and FDA regulations. Another issue is the stability of your material, both for shipping and application.

We founded SteriCoat (now Semprus BioSciences) to explore the biomedical applications of AMPs. We were thinking we could coat devices covalently with AMPS, and reduce risk for catheter infections. However, we ended up turning to a totally different technological solution. The regulatory path drives a great deal of your timeline, risk, and funding requirements. Incorporating an agent that the FDA may consider an active agent would raise the burden for the company. Whether or not this decision is made by the FDA or whether or not this is the correct decision, this raises your risk. We pursued a totally synthetic chemistry that was less likely to be viewed as "active".

We had overcome many challenges with the AMPs themselves along the way. My PhD project was AMP design and application. We initially were looking at creating in vitro translation systems to avoid killing cells. I worked in a metabolic engineering lab, and we did microbial screens too. Early in my PhD years, we had a computational tool for AMP design, and we wanted to screen a bunch of them. The problem is that it kills off your bugs. Can you grow it with an inactive form, or use an excretion tag? Can we use a bug that isn't targeted by the AMP? The cleverest thing we found was designing them to be expressed with an inducer. We used parallel stamps, one with an inducer, one without, and we looked at the zone of inhibition. Basically, we used replica plating to identify the most efficient producers of AMP.

Then, chemical synthesis of AMPs became really inexpensive! You could look into proteins that are difficult to synthesize in-vitro, for example defensins, and there are databases of these.

iGEM: How did you decide to become an entrepreneur?

CL: I came into grad school wanting to do something entrepreneurial in the Langer lab and really make something happen. What really got me going was when we started to get good results, Langer put me in touch with David Lucchino at the Sloan school, and we created a business plan that won competitions at MIT, Harvard, Oxford, Cambridge, and other universities. We started gaining momentum, and things got very interesting. When you're an entrepreneur, you're also a fund raiser, a writer, and a team leader, regardless of the academic field or industry. Of course its risky, but everything's risky. In the end, you get the skills that will make you flexible and valuable regardless of what you achieve.

We thought about applications as diverse as naval ships, water processing plants, orthopedics, and food safety. A lot of the decision making for our strategy came down to cost and timeline for development, market size and margin for the product, and the duration of stability and activity required in a given application.

iGEM: How do you translate cool science into something viable?

CL: You have to go from a vision to the unmet need. We eventually licensed a technology I didn't invent, and that's okay. Many large companies suffer from, "Not Invented Here" bias, but as a small company, you need to put your ego to the side and lead the team to the best solution.

"Many large companies suffer from, "Not Invented Here" bias, but as a small company, you need to put your ego to the side and lead the team to the best solution."

You also have to get technologists to think about the business issues. How does your technology address the business concerns and give you an advantage in the market? You say: This is how we're going to exploit this unmet need through business. Here's the unmet need, here's our solution, here's why we'll be the only ones to produce it, and here's how we're going to execute.

As one of my fellow board members said: "I was a scientist and now I'm a businessman." I think academia is about branching out and looking forward, but business is about having a specific target and finding the shortest path. So it's a very different mindset, but the business mentality is a learnable skill.

iGEM: Did you consult experts on the non-scientific aspects?

CL: Startups are brutal. You can't spend all of your money on lawyers and attorneys. You really have to make yourself an expert. You need to know the science, and you can't afford to have consultants do all of the work for you, so you need to do most of the legwork.

Once you generate specific questions, you can get a lot of value from an hour-long meeting with an expert: "here are the critical things I saw, and here's what I'm confused about." That's the best way to be efficient.

I got to help do a license, manage IP strategy, and design a clinical trial. There is enormous potential to learn about a wide variety of fields in a new start-up. You don't have to be the expert in each area, but if you can ask the hard questions at the interfaces and can get the right advisers, then you can be a successful leader.

iGEM: What about intellectual property issues?

CL: When I was the Chief Technology Officer, I was doing all of the pitches as well as managing the Intellectual Property portfolio.

I spent a year understanding 500-1000 papers or patents in the field, and we had to identify key differences between their ideas and ours. The space is busy, but then again most valuable spaces are busy and it is the entrepreneurs responsibility to carve out their space.

iGEM: Are there other ways to use AMPS? What recommendations would you give to our team going forward?

CL: To start off, you should think about peptides or proteins that are bigger than what is chemically synthesizable. What would be cool to make a surface for? What could you want to do with a protein bound to a surface? But you can't just keep brainstorming about the technology, you really need an unmet need to pull you in the right direction.

"...you can't just keep brainstorming about the technology, you really need an unmet need to pull you in the right direction."

The first thing my cofounder did was drag me to the hospital and get me to start talking to the nurses. I had to become an investigative reporter, an engineer asking questions. But I had to do it, because often the unmet need is outside your area of expertise.

I'd also think hard about "why microbial production"? When would your peptides be cheaper? Are they super-complex peptides that you couldn't produce otherwise? Are there powerful ways to use the bacteria as a living system? From a commercial perspective, think really hard about the unmet need that only you can address.

Over the summer, Yale iGEM member Ariel Hernandez-Leyva delivered a class for local high school students entitled:
New Pills for Old Ills: Antimicrobial Peptides and a Possible New Approach to Molecular Medicine
The course started by focusing on the history of antibiotics and anti-microbials and moved on to discuss the current issues with antimicrobial resistance. A possible solution to this process, anti-microbial peptides, was discussed and the biochemistry behind the peptides was explained. To conclude, we talked about how the science of synthetic biology could fit into an era of molecular medicine through a discussion of the production of anti-microbial peptides in bacteria for medical use.