General Concern and Regulation of Biosafety
All experiments of our iGEM project were conducted in the Biosafety Level-1 laboratories under the regulation of Environmental Safety and Health Center of Chung Shan Medical University. The emergency exits and basic risk assessment are the first things to check everyday prior to experiments.
All members of the laboratory were well-trained by the mentors and had ability to conduct experiments in a safe environment. People who entered the laboratory should follow the established laboratory safety principles of our school. Master’s degree students of the lab and our instructor helped us to complete the safety training.
Material Safety Data Sheet, MSDS, including every chemical, reagents of the lab are printed and collected in a data folder. The folder is placed in a significant place near the door, which is easy to find and read in case of emergency.
All kind of waste produced in the working area of the laboratory are considered biohazard waste, including every experiment and consumptive material. Those biohazard wastes would be collected in a specially labelled red plastic bag and sent to Environmental Safety and Health Center of Chung Shan Medical University for the further disposing process.
Legal Compliance Implemented in the Project Design
Our team want to turn the aflatoxin-degrading enzymatic antidote into a real product, and the design has been changing along with our entrepreneurship research, integrated human practice for new safety concepts. In spite of the fact that safety issues were put into concern, a product should still comply with the local laws and regulations. We reviewed the laws and regulations related to GM products of Republic of China, and summarized the principles of GMO food application (exclusively for GM microorganism):
1. Safety assessments must be done in the product development, and it should include the following items
A. Compositional analyses of key components
B. Toxicity and sensitization of the expressed material
C. Safety assessment of its metabolites
D. Changes in nutrition facts
E. Antibody resistance and its gene transduction
F. Viability and residence of the GM microorganism in the gastrointestinal (GI) system.
2 Self-cloning is a better choice. The inserted genetic material may be made synthetically, but it must better come from an organism of the same or closely related species.
3. Strains with safe consumption histories are the basis of developing genetically modified abiological foods.
4. GMO products should meet GRAS standards
5. All GMO products should be approved by FDA before being released to the market.
Reviewing the antidote project, using yeast as the host for the insert gene is the best choice for us. The selection marker or the newly constructed vector is non-antibody-resistance, and the cell was lysed in the manufacturing process indicating that there is no need to evaluate the viability and residence of the GM microorganism in the GI system. Moreover, yeast is a GRAS organism since there has been a long history for human beings eating them. Those mentioned above are the primary consideration when we designed the product. However, changes in nutrition toxicity and sensitization of the expressed material were not examined due to the time and financial limitations, and those would become the future work that we are going to study.
Safety Considerations and Project Evolution Integrated with Human Practice
The following text explains the initial ideal of our aflatoxin-degrading enzymatic drug and its evolution with safety concerns. With the feedback from Integrated Human Practice and Public Engagement, safety issues have driven the appearance, type of content and the host organism to transform. Let’s see the story behind our project!
Our project aims to mass-produce an enzymatic drug that could degrade aflatoxin inside our body. Since we are going to engineer something that would enter our body, the safety issue of this project is a very serious concern and it propels our project to evolve.
At the very initial phase of our brainstorming, we came up with these ideas below：
1. The enzyme should have the ability to degrade aflatoxin.
2. The enzyme should work well at 37℃ (body temperature).
3. The enzyme should not be reported as a toxic protein in the previous research.
4. The enzyme should not work in our circulation system for the sake of immune problems.
5. The enzyme should be produced in a cost-effective way
Why a capsule?
After choosing the specific functional enzyme from previous researches, we came up with the first prototype of this drug, and it fits all characteristic above. The drug was designed as a “capsule” for the sake of safety, and it contained the whole microorganism inside to lower the price. It is common sense that most drugs would be more effective through intravenous injection (IV). However, foreign protein or peptide in blood would have a great chance to bring severe immune response to the patient, indicating that the disadvantages might outweigh the advantages. Hence, we want to create an oral drug that would not directly enter our circulation system, and that is the reason why the drug in our project is a “capsule”.
To lower the cost of producing such kind of enzymatic drug, we planned to put the whole microorganism in the capsule in order to prevent protein purification, which was the most costly step in the manufacturing process. Nevertheless, eating genetically modified (GM) microorganism might lead to some safety problems, and we noticed that it was not persuasive enough for people to accept this kind of product via our public engagement. Thus, choosing a suitable vector and host that produce the enzyme became an important issue in our project.
Setting the ideal properties of the capsule
Our team further set that the ideal content of the capsule should meet the following criteria:
1. The host should be dead and they would no longer be able to reproduce in our body or the outside environment.
2. The cytoplasm of the host should be safe and toxin-free.
3. The product should comply with the law of conduct.
4. The product should be Generally Recognized as Safe (GRAS)
E.coli was just a temporary protein-producing platform for research in our project. Although E.coli was common and easy to culture, it was not the ideal host at all. The major reason we did not want to put E.coli in our capsule was that E.coli contained some endotoxin, and it was not generally acceptable.
At the time we successfully purified the enzyme MSMEG5998 and Thioredoxin-MSMEG5998 fusion protein from E.coli and proved their degrading ability, we began to seek for a suitable vector and host to produce the enzyme.To search for a suitable vector and host, our team went to visit several specialists and asked for their opinions.
Choosing GRAS/Food grade heterologous protein production vector/ host
In August, we visited professor Yeh, C. M at National Chung Hsing University, the chairperson of “GRAS/Food Grade Heterologous Protein Production Platform”, and shared our idea about cloning the enzyme-producing gene into a suitable vector and host. Fortunately, she invited us to participate in the forum of that platform, which was the largest annual event of industrial fermentation, and we learned a lot from it.
The ultimate goal of the heterologous protein production industry is “Good host produces good protein”. Genetically modified (GM) product does not equal to unsafe products, but safety issues must be noticed prior to everything. According to “Guideline for the Conduct of Food Safety Assessment of Foods Produced Using Recombinant-DNA Microorganisms” from United Nations (UN), a safe host must be generally recognized as safe (GRAS) or food grade. In 2003, Codex Alimentarius Commission of UN suggested that food grade hosts should not use antibody resistance genes as selection markers. Although food and medical applications of GM microorganism are still at a starting stage, regulators are attempted to think ahead and standardized the rule of the whole industry. Our team wanted to create a product that is safe enough for human to eat, thus we chose Lactobacillus as our host to produce the recombinant enzyme we constructed (Thioredoxin-MSMEG5998).
From Lactobacillus to Yeast
After deciding to use Lactobacillus as our host organism, we started to study how to construct an expression vector that is safe with high efficiency. We met local Lactobacillus experts such as professor Yeh, C. M, Dr. Chen, P. H. to discuss our idea.
Lactobacillus was great. People had been eating them for a long time throughout history, and the safety of their cytoplasm are guaranteed. Furthermore, in many cases, the recombinant protein tends to form inclusion bodies in E.coli, while Lactobacillus does not. Nonetheless, there are several drawbacks:
1. The copy number of Lactobacillus plasmids is usually low, resulting in a low production rate.
2. Safe vectors using non-antibody-resistance selection markers are still hard to find, and most of them are patented.
3. Expression system of Lactobacillus is complicated, leading to a higher construction failure rate.
To overcome the difficulties, we went to Food Industry Research and Development Institute (FIRDI), the largest central food industry research organization in our country, to visit Mr. Liao, C. C. and his research team. Mr. Liao was the director of FIRDI, and he was also one of the most remarkable expert in Lactobacillus research.
Although Lactobacillus sounds rather perfect, it is still controversial since food and drug industry applications of Lactobacillus remain unrecognized. And the high construction failure rate made it a big problem for our team for the sake of time limitation. However, yeast tends to be much better in this project, as they had wide application in bread, beer and industrial heterologous protein production. Mr. Liao suggested us to focus on making a minimum viable product (MVP) in this iGEM project in order to realize the idea with the minimum amount of time. Hence, our project evolved to the final version: using yeast as our host of recombinant enzyme gene.
A safer design-non-antibody-resistance selection marker vector
At the end of August, we went to visit the R&D team of Sinphar Group, one of the largest pharmaceutical factories in Taiwan. This visit helped us to further understand the pre-marketing risk assessment, safety concerns and difficulties of an enzymatic drug development. According to the pharmaceutical professionals’ statement, a drug or health-care food product should do minimum harm to our body. And it strengthened our determination towards choosing a safe vector to carry our aflatoxin-degrading enzyme gene.
Yeast-the final host of our project
Saccharomyces cerevisiae, a kind of common yeast strain, became the ultimate host for the enzyme in our project. The cytoplasm of S. cerevisiae is safe, for human beings have been eating them for thousands of years in bread and other food. The newly constructed vector used certain amino acids as selection markers, indicating that there were no antibody-resistance problems in yeast. In addition, yeast was much easier to culture in the laboratory. The above reasons is why S. cerevisiae was chosen as ideal host of the aflatoxin-degrading enzyme gene.
General Concern and Regulation of Biosafety
Legal Compliance Implemented in the Project Design