Safety
Describe all the safety issues of your project.
Describe all the safety issues of your project.
Our ultimate goal is to create a de novo bacteriophage-based vaccine for animal and human health. Animal studies are inevitable to verify the effectiveness of such a vaccine. And the application of animal use reviewed by an Institutional Animal Care and Use Committee (IACUC) is labor-, time- and money-consuming, especially for a short season (less than a year) and budget limitation in an iGEM project. Therefore, for the Safety issues, it’s definitely challenging and caused us to rethink how we could achieve a Proof of Concept to demonstrate our project. As the abstract schematic illustration below, we clearly dissected our project into two separate and independent small specific aims, and fit all into safer way for a proof of concept before animal use approval:
Building up phage vaccine model including phage strain, phage engineering, host bacteria isolates, antigen production, antigen purification, etc.
Implementation in an animal model. Before the approval of animal study, we provided two kinds of evidences to support our concept.
In the beginning, we would like to harvest and isolate bacteriophages from the environment for infection of the commensal E. coli. After doing HP activities and consulting experts in GMO including phage engineering, we discarded the idea of collecting environmental phages which could possibly harbors virulent genes. Instead, we decided using one of lab model bacteriophages, T7 phage1, and tested the T7 phage susceptibility of the commensal E. coli2.
Secondly, to engineer the phage, at first, we want to use the skill developed by our previous iGEM team (i.e., iGEM Team Mingdao in 2021), who has utilized Tol2 transposase-mediated gene editing on the phage genome. The transposable elements may result in Gene Drive issue to generate a possible trait to offspring although in very limited possibilities. Finally, we used a restriction enzyme system provided in the Novagen® T7Select® 415-1 Cloning Kit (Merck Millipore) to engineer our T7 phages.
Lastly, although we learned how to isolate the mouse intestinal commensal E. coli from fresh feces, the E. coli isolates may contain possible pathogenic virulent features3, even collected from the mice reared in the individually ventilated cage (IVC) in the animal center. We decided using the commensal E. coli isolates, which were collected and identified by Prof. Ming-Shiou Jan’s laboratory at University of Chung Shan Medical University several years ago. The isolated E. coli strain was being used in many studies and verified without major known virulent genes3.
For a vaccine development, most of people think about the Spike protein of SARS-CoV-2. So did we in the beginning. We did some researches and found the possible physiological toxicity of the Spike protein including disrupting lipid metabolism in liver, heart, kidney damages4, and even crossing the blood-brain barrier in mice5. The safety of the Spike proteins is concerned6.
We finally chose a model antigen, ovalbumin, to be engineered for the demonstration of our phage vaccine system. The chicken ovalbumin (OVA) is a glycoprotein, which is a major component of chicken egg whites, and harbors immunogenic properties in vaccination experiments7. The recombinant OVA protein is readily purified in E. coli BL21 system driven by T7 promoter and triggered by IPTG induction. Therefore, it’s considered as one of the model antigens used to study immune responses in animal models.
Before the approval of animal use application, we provided supporting evidence from the researcher’s lab and the published paper, as well as give examples of using phage – commensal bacteria – protein production model as the same approach in an iGEM project and a pre-clinical trial of a biotech company.
Prof. Ming-Shiou Jan’s laboratory at University of Chung Shan Medical University observed a phenomenon that the lysates of phage-infected bacteria have an immunostimulatory property like adjuvant, which has been confirmed in the published paper by Zhu J, et. al. for phage particles8 and by Lim J, et. al. for bacterial envelope9. The results encouraged us to design a synthetic biology approach to create phage vaccine in our project.
In the previous iGEM project of TUDelft in 2020, they aimed to kill locusts by the toxic proteins produced by the gut bacteria which were infected with the engineered phages carrying toxin gene (Cry7Ca1, a gene from Bacillus thuringiensis) through the spray. Their mode of phage - gut commensal bacteria - protein production encouraged us to design in a similar way to create phage vaccine in a relationship of phage, intestine commensal E. coli and antigen protein production.
For clinical application, an Israel biotech company, BiomX, Inc., has completed a preclinical study demonstrating that a feasible approach to cure colorectal cancer in mice by cytokines production in bacteria surrounding the tumor through the infection of the engineered phages carrying genes of GM-CSF, cytosine deaminase, IL-15 in a similar mode of phage - tumor co-existing bacteria - cytokine protein production. The success of the trials gives us hope to believe that a potential phage vaccine can be created in such a way with synthetic biology.
Prof. Jan helped us apply for the animal use. And, the form of Animal Use has been submitted to the reviewers in Institutional Animal Care and Use Committee (IACUC) of University of Chung Shan Medical University. We’re waiting the approval. (Go to check the application form in Chinese version)
We cultured bacteria of E. coli DH5α and BL21 (DE3) strains in a laminar flow clean bench throughout the whole process of gene cloning and protein expression/purification.
As for the mouse intestinal commensal E. coli at BioSafety Level 2, we performed all of the related experiments at P2 laboratory of Prof. Ming-Shiou Jan at Chung Shan Medical University and conducted the experiments under the supervision of him or his doctoral students.
The T7 phages we handled are considered as BioSafety Level 1 by BCRC in Taiwan and a phage expert, Prof. Chih-Hsin Hung of I-Shou University. We engineered phage genome by Novagen® T7Select® 415-1 Cloning Kit (Merck Millipore).
All chassis we used won't pose a threat even if they escape from the lab. These chassis can't directly cause any disease to humans under general circumstances and present minimal hazards to the environment. Our engineered phages might enter the environment accidentally. As mentioned, the product is only used in the lab, therefore, just as doing on E. coli and recombinant DNA, the regulation is monitored by the safety committee and the government.
Finally, all of the waste from the lab was disinfected by an autoclave sterilizer and transported away by a local lab waste management company.
We have our own biosafety committee, which consists of two research teachers. They oversee proper work area conditions by checking on disposal of Petri dishes and liquid wastes, sanitation, and teaching proper laboratory techniques. Our guidelines, taken from bio-risk management posts on the website of CDC Taiwan, cover lab safety policies and procedures ranging from lab-specific rules to behavior. For example, we prohibit food, open-toed shoes, and drinks in the lab. We also have a thorough clean-up procedure. For example, we have waste bins for used tips, which are autoclaved before disposal, and liquid wastes are bleached. Teachers acquaint us with all experiment techniques. Moreover, he is also highly familiar with iGEM owing to years of experience as the instructor of the iGEM team of our school.
The main regulations regarding bio-safety and bio-security, including biosafety inspections of high-containment laboratories, biosafety technical specifications and guidelines, biosafety education and training, bio-risk management post on the website of CDC Taiwan. See the guide book and the related documents here.
We’ve received related training on topics as follows.
All personnel received a tour around the lab and were informed of the rules upon entering the lab, the main rules include: