Introduction

Our project’s focus was on the development of engineered bacterial strains which could act as vaccine delivery systems by expressing recombinant genes. Our long term goal would be to have a positive impact of on the aquaculture and fisheries industries, more specifically, the high end and mid level ornamental fish industry which breed varieties of koi fish (Cyprinus rubrofuscus). Our final engineered designs we worked so hard on were designed to be capable of providing these high value fish varieties immunity against the Cyprinid herpes virus-3, which cases the high mortality rate disease Koi herpesvirus disease (KHVD).

As always the design of a genetically engineered system requires many revisions, multiple identification of new problems, and several development of potential solutions all of which needed to be informed by good science and have logical reasoning for pursuing. In this sense, each stage of our project since the early team meetings of our BioBuilder club have been informed by the individuals and organizations we sought out and contacted, in order to ensure we had a safe, consequential, interesting, and scientifically sound project. This section outlines our efforts in that endeavor.

The goals of our integrated Human Practices work can be summarized in the following points:

1. 1. We wanted to first learn what were the issues associated with the koi fish industry and more specifically to see what a potential problems exists that could be solved, or at least improved, with synthetic biology concepts. This was our generating ideas and identifying problems part of the engineering design process.

2. 2. We then wanted advice on the early stages of our genetically engineered systems. Here we sought academic experts that have worked on the health of fish in aquaculture and precisely the health and breeding of Koi. The ideas generated we had at this stage were based on discussion among ourselves and from a review of the associated literature. We were able to discuss in detail our potential solutions with professors and bring back more ideas for the refinement stage of our project design.

3. 3. When we asked Mr. Wang if they vaccinate the fish against any disease, the answer was no. The Koi the school purchased were the cheap ones, so they think the fish food that containing the immune system boost ingredient will be enough for our Koi. The fish themselves are also not vaccinated against anything prior to purchasing. We left this meeting with clearer objective and our project progressed from simply developing a system to help provide a boost to fish health through the production of adjuvants in fish food to one where we wanted to develop a specific vaccine for a specific pathogen that is a common killer I our part of the world (East China). We also had the objective of starting to look for people to learn from that are involved in the high level Koi breeding.

Koi pond inside our school｜May 17th, 2019

Identifying Problems & Developing Solutions.

Our iGEM project inspiration came from one chance event at our own school’s Koi pond. The first person we decided would be a great resource of information about potential problems we could identify begin to develop synthetic biology solutions to was our school head of operations Mr. Wang. We reached out to him early in our project developing as we felt he could give us the first-hand information about Koi breeding and health issues associated with them. We asked Mr. Wang specifically about the pond maintenance, the relevant cost of running the pond and of raising the (such as cost of fish food and drugs),. We also asked about the mortality rate of fish and whether the fish are getting any kind of treatments to help their health or indeed even on a vaccination program.

According to Mr. Wang, they feed fish every morning with fish food that containing amylose and lecithin in the ingredients that will boost the fish immune system. They also clean the pond every 2-3 months to keep the water clean, tgis was after the early years when the mortality rate of fish was high, but importunely he could not tell us what exact disease the fish were getting. In the first few years, the school spends 3000-4000 RMB per year on purchasing new fish due needed to replace the fish which die throughout the year. According to Mr. Wang, in 2017 the mortality rate of the fish was very high that they had to spend almost 3 times money on purchasing new fish than regular years. Again it was unclear what disease caused this problem as Mr. Wang tells told us the koi they buy is at the lower end of the market and are very common so are not the sought after expensive breeds.

When we asked Mr. Wang if they vaccinate the fish against any disease, the answer was no. The Koi the school purchased were the cheap ones, so they think the fish food that containing the immune system boost ingredient will be enough for our Koi. The fish themselves are also not vaccinated against anything prior to purchasing. We left this meeting with clearer objective and our project progressed from simply developing a system to help provide a boost to fish health through the production of adjuvants in fish food to one where we wanted to develop a specific vaccine for a specific pathogen that is a common killer I our part of the world (East China). We also had the objective of starting to look for people to learn from that are involved in the high level Koi breeding

Ocean University and national Pathogen Collection Center For Aquatic Animals｜July 17th, 2019

Design and Refinement.

After talking to Mr. Wang, learning more about Koi fish breeding, and most importantly develop a clearer objective, we got back to group discussion and literature studies which helps us develop some possible solutions. It was time for the SUIS Shanghai IGEM to seek out academics to help us with choosing and refining the best approach so we could achieve our overall project aim. We therefore planned a meeting with Professor Chen Zai Zhong and Professor Gao Jian Zhong at Shanghai Ocean University with the main purpose of presenting to them our projects aim and the designs we were developing at that time. We also had the secondary purpose of going to visit the universities off campus Koi fish breeding farm. Professor Chen has many years of experience in ornamental fish breeding and Professor Gao is specialized in prevention and control of aquatic animal diseases. The Shanghai Ocean University was a fantastic choice for us as it housed the National Pathogen Collection Center for Aquatic Animals near the school of veterinary where lots of active research on pathogens to aquatic animals and their DNA was performed.

The day long trip was very productive and we must thank the two professors and their faculty members for their enthusiasm and extremely valuable insights they provided to us. We very quickly were able to choose the pathogen we wanted to targets for our vaccination during this meeting. Originally, based on the literature we had a few candidates for our project including: Myxobolous sp., Goussia carpelli and, Cyprinid herpes virus-3 . We were informed that the third option of the three was the best choice for developing a vaccine based on global impact and the fact that the Cyprinid herpes virus-3 had its genome sequenced. This virus causes devastating effects to the industry as if one Koi is infected, the entire pond needs to be culled. The professors told us a story of how the entire Koi breeding program was destroyed in Israel because of it. During our visit we also go to view their fish farm and saw the machinery they use for vaccination of the fish. This machine consisted of a network of tubes that the fish can swim through and get dozens of pinprick injections along the way which is relatively painless and stress free. This machine however was only one of a few in the world so when we discussed our plan of oral vaccination due to wanting to reduce handling of fish, the professors thought it was a good idea but warned of a drop off in vaccine effectiveness with oral administration compared to injections.

Both professors were deeply engaged in our discussion and very interested in our ideas. We were received very well and obtained some great insights into future challenges we will face. Professor Gao expressed concern toward the availability of synthesizing antibodies inside the fish gut. Gut is a complicated system where many different types of bacteria coexist, so they cannot be sure if specific antibodies can be expressed and the exact amount will they be expressed in gut. Professor Chen also pointed out that despite the model of iron Qs system can theoretically express target protein, but there are other gene expressions happened in the gut that may disturb the synthesis of antibodies. We left our whole day meeting having developed our project design much further than anticipated. A summary is given below:

• - We learned that oral vaccination through mixing with fish food can be effective for large scale vaccination. Booster doses may be required from time to time however

• - We were able to choose the target pathogen for the development of our vaccine based on the availability of genes, the amount of previous research into this particular virus and the historical and economic impact associated with outbreaks of Koi herpes virus diseases (caused by the virus). This target would give us the best chance to have a positive impact on the koi fish industry and would garner interest from the people within it.

• - We discussed at length the approaches we would make to develop our vaccine. Plasmid DNA vaccines are tightly regulated and it was recommended that we not pursue this area. Nor was it recommended we use partially attenuated whole virus or virus coats again due to regulations and the fact that we are just a high school. The idea of bacterial vaccines expressing recombinant proteins as antigens was attractive to the professors as they were aware of such research and after learning more about what iGEM was they agreed with us that this seems the most relevant course of action.

• - We also got good feedback on the idea of using a lactic acid bacteria as our delivery choice as we were told that many hold generally regarded as safe (GRAS) status and can be easy to work with.

• - The questions and problems remained as to how we could protect any antigen we use through the alimentary canal of fish. Bioencapsulation and the use of inducible promoters were discussed as possible solutions. When iron starvation was discussed the professors indicated that they thought it was possible but there will be a lot of metabolite “noise” in the gut environment and the best advice they could give here was that it needed to be tested and see.

Shanghai International Aquaculture Exhibition｜August 28th, 2019

After completing the design of the experiments, we want to know more about Koi breeding from the Koi fish market to decide the best way of vaccination.

We found out that the Shanghai International Aquaculture Exhibition was going to be held in late August, where all business sectors that related to aquatic product will attend. It would be the perfect place for us to reach to Koi fish breeding companies. We've been able to talk to many people that own Koi fish farms and ask them specifically about the most effective method of vaccination for different scale fish and which method they prefer to use the most.

According to them, as Koi can live for over 70 years and sell for very high price in the market, they spend big amout of money in keeping the Koi healthy every year, and vaccine always plays an important role in it. To appropriately give the vaccine, timing and method of vaccination also need to be considered. They suggested making oral vaccine for their low cost and high efficiency. Unlike injection, which may lend pressure or physically harm the fishes, oral vaccination is also the safest compare to other methods.

We are also very lucky that one of the fish drug company showed strong interests in our recombinant bacteria idea and willing to talk to us deeply in the future on making the vaccine into real sample once we proved our experiment worked as although they have been working on making fish drug and vaccine for many years, our idea of using a promoter to control the expression of the antigen with live attenuated vaccine-carriers is still brand new and full of potential.