Safety

Describe all the safety issues of your project.


OUR GOAL

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:


Aim #1:

Building up phage vaccine model including phage strain, phage engineering, host bacteria isolates, antigen production, antigen purification, etc.


Aim #2:

Implementation in an animal model. Before the approval of animal study, we provided two kinds of evidences to support our concept.

  1. providing a verified data or observations from other lab work conducted previously, in which the mixture of purified antigen proteins (i.e., ovalbumin in our case) and WT phage-infected and killed bacteria (i.e., the intestinal E. coli in our case) can induce immune responses in mice. Such a mixture can be considered as a similar or even the same product we supposed our engineered phages do through transferring genes into the host and producing protein of interest in the host as demonstrated in the Aim #1.
  2. giving lab or clinical examples of the same idea of using phage – commensal bacteria – protein production. Here are an iGEM 2020 project by of TUDelft and an Israel biotech company, BiomX, Inc., respectively.

SAFE PROJECT DESIGN

Choosing a non-pathogenic chassis and a safer engineering method

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.

Choosing parts that will not harm humans / animals / plants

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.

Providing supporting evidences for a proof of concept

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.

1. Supporting evidence from Prof. Jan’s lab

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.

2. iGEM 2020 project - TUDelft

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.

3. Clinical trial - BiomX, Inc

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.

Animal study form and application

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)


SAFE LAB WORK

Organisms

Biosafety Level 1

  • E. coli K-12 DH5α, E. coli BL21 (DE3) are common commercially available strains.
  • T7 bacteriophage is a common laboratory model organism.

Biosafety Level 2

  • Mouse intestinal commensal E. coli were harvested from lab BALB/c mice feces and isolated by Prof. Jan’s lab.
  • We used the isolated E. coli strains for testing their susceptibility to T7 phages.
  • We’ve filled in the Check-In Form for these isolated E. coli strains.
  • We conducted experiments with the related materials in Prof. Jan’s lab at Biosafety Level 2 and under the supervision of Prof. Jan or his PhD student.

Novel BioBrick Parts

Part: BBa_K4150000

  • Name: g10.RBS
  • Function: the enhanced version of the canonical RBS
  • Source: synthesized by IDT
  • Usage: to increase protein production in E. coli
  • Species: T7 bacteriophage gene 10 (g10) leader sequence
  • Risk group: Biosafety Level 1

Part: BBa_K4150004

  • Name: His-OVA
  • Function: ovalbumin protein is a major component of chicken egg whites
  • Source: codon optimized in E. coli and synthesized by IDT
  • Usage: to act as a antigen for vaccination test
  • Species: Gallus gallus
  • Risk group: N/A

Risks Management

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.

Biosafety Committee

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.

Guidance of Risk Management

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.

Safety Training

We’ve received related training on topics as follows.

  • Lab access and rules
  • Responsible individuals
  • Differences between biosafety levels
  • Biosafety equipment (such as biosafety cabinets)
  • Good microbial techniques (such as lab practices)
  • Disinfection and sterilization
  • Emergency procedures
  • Transport rules
  • Physical biosecurity
  • Personnel biosecurity
  • Dual-use and experiments of concern
  • Data biosecurity
  • Chemicals, fire and electrical safety

Work Areas

  • Open bench
  • Biosafety cabinet

Risk Management Tools

  • Accident reporting procedure including emergency phone number and the instructor who is in charge.
  • Personal protective equipment including lab coats, gloves, eye protection, etc.

Safety Regulations

All personnel received a tour around the lab and were informed of the rules upon entering the lab, the main rules include:

  • No running or sudden movements in the lab.
  • No eating in the lab.
  • All experiment waste must be thrown in a designated bin, which will then be put through a machine for sterilization.
  • All pipettes must be sanitized after use.
  • All equipment used must follow the user manual.
  • Experiments can only be performed with gloves and lab coats on.
  • All personnel must wash their hand upon entry into the laboratory and wear suitable clothing.

Safety Equipment Photo

  • Safety equipment I: eye wash fountain
  • Safety equipment II: emergency shower
  • Laminar flow hood – recirculating clean bench
  • Autoclave sterilizer

REFERENCE

  1. Yue H, Li Y, Yang M, Mao C. T7 Phage as an Emerging Nanobiomaterial with Genetically Tunable Target Specificity. Adv Sci (Weinh). 2022 Feb;9(4):e2103645. doi: 10.1002/advs.202103645. Epub 2021 Dec 16. PMID: 34914854; PMCID: PMC8811829.
  2. Kasman LM. Barriers to coliphage infection of commensal intestinal flora of laboratory mice. Virol J. 2005 Apr 15;2:34. doi: 10.1186/1743-422X-2-34. PMID: 15833115; PMCID: PMC1097760.
  3. Pakbin B, Brück WM, Rossen JWA. Virulence Factors of Enteric Pathogenic Escherichia coli: A Review. Int J Mol Sci. 2021 Sep 14;22(18):9922. doi: 10.3390/ijms22189922. PMID: 34576083; PMCID: PMC8468683.
  4. Nguyen V, Zhang Y, Gao C, Cao X, Tian Y, Carver W, Kiaris H, Cui T, Tan W. The Spike Protein of SARS-CoV-2 Impairs Lipid Metabolism and Increases Susceptibility to Lipotoxicity: Implication for a Role of Nrf2. Cells. 2022 Jun 14;11(12):1916. doi: 10.3390/cells11121916. PMID: 35741045; PMCID: PMC9221434.
  5. Rhea EM, Logsdon AF, Hansen KM, Williams LM, Reed MJ, Baumann KK, Holden SJ, Raber J, Banks WA, Erickson MA. The S1 protein of SARS-CoV-2 crosses the blood-brain barrier in mice. Nat Neurosci. 2021 Mar;24(3):368-378. doi: 10.1038/s41593-020-00771-8. Epub 2020 Dec 16. PMID: 33328624; PMCID: PMC8793077.
  6. Theoharides TC, Conti P. Be aware of SARS-CoV-2 spike protein: There is more than meets the eye. J Biol Regul Homeost Agents. 2021 May-Jun;35(3):833-838. doi: 10.23812/THEO_EDIT_3_21. PMID: 34100279.
  7. Geary TW, Reeves JJ. Production of a genetically engineered inhibin vaccine. Vaccine. 1996 Sep;14(13):1273-9. doi: 10.1016/s0264-410x(96)00014-x. PMID: 8961517.
  8. Zhu J, Jain S, Sha J, Batra H, Ananthaswamy N, Kilgore PB, Hendrix EK, Hosakote YM, Wu X, Olano JP, Kayode A, Galindo CL, Banga S, Drelich A, Tat V, Tseng CK, Chopra AK, Rao VB. A Bacteriophage-Based, Highly Efficacious, Needle- and Adjuvant-Free, Mucosal COVID-19 Vaccine. mBio. 2022 Aug 30;13(4):e0182222. doi: 10.1128/mbio.01822-22. Epub 2022 Jul 28. PMID: 35900097; PMCID: PMC9426593.
  9. Lim J, Koh VHQ, Cho SSL, Periaswamy B, Choi DPS, Vacca M, De Sessions PF, Kudela P, Lubitz W, Pastorin G, Alonso S. Harnessing the Immunomodulatory Properties of Bacterial Ghosts to Boost the Anti-mycobacterial Protective Immunity. Front Immunol. 2019 Nov 22;10:2737. doi: 10.3389/fimmu.2019.02737. PMID: 31824511; PMCID: PMC6883722.