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. Once we developed our idea and design to an advanced stage we wanted to seek out people within the fish feed and drug industry, as these people are also experts in the final product for therapeutics relating to aquaculture. At this stage we had identified new problems that needed overcoming and got valuable insight from these people on the methods used to deliver such medicines and biomolecules to fish. We also gained insight into the business side of things and how we could possibly eventually develop our idea into a marketable product should any trails we performed prove successful.

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.

Ocean University｜July 17th, 2019

After talking to Mr. wang and knowing more about Koi breeding, SUIS Shanghai IGEM team planned a meeting with Professor Chen Zai Zhong and Professor Gao Jian Zhong at Shanghai Ocean University to discuss our project design. Professor Chen has many years of experience in ornamental fish breeding and Professor Gao is specialized in prevention and control of aquatic animal diseases.

(Although both professors expressed strong interest in our project, 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. Given that only forms of amino acid can be taken in into gut and amino acid does not have antigenicity. How to synthesis giant protein that can be identified by white cells and triggered the immune responses is a problem to solve. 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.) (No idea)

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.