Project Safety

Laboratory Safety


Safety training on handling hazardous materials, emergency protocols, and responsible disposal of biohazardous waste was conducted before starting working in the lab in May 2023 by Lab Assistants of the NU Biology department. It's this commitment that allowed our iGEM team to maintain the highest standards of science. The laboratory work was conducted from May to October 2023 under the supervision of specialists from the School of Social Sciences and Humanities.
The laboratory in which we worked is 7.410 on the Nazarbayev University campus and is considered a BSL-2 level laboratory. Below you can find photos of our laboratory and its equipment. Also below you can find guidelines with the basic requirements for levels BSL-1 and BSL-2.
Figure 1. Laboratory photographs

Safety in our Project



On June 12, 2023, all wet-lab and dry-lab members had a lab safety trainins, and since then the experimental part of our iGEM team began, and during the experiments, various precautionary and safety methods were used when working with reagents, bacteria, and cancer cultures.
In addition to the basic necessary safety measures, such as Personal Protective Equipment, working under the Hood, etc., our team followed the principle of never working alone and always informing lab coordinators working in the laboratory about our actions, especially when it comes to potentially hazardous materials
Among all the materials, during our work, there were, of course, those that required special control.
E. Coli DH5-alpha and BL-21 non-pathological strains were used for plasmid multiplication and protein purification. These bacteria are not dangerous and fit the BSL-1 level, which requires us to work under Bacterial Hood and the mandatory use of PPE2.
Ethidium bromide, a widely used marker in gel electrophoresis experiments, requires special conditions for storage and use, as it is also toxic and a potential mutagen that can be easily absorbed through the skin. Our team used all precautions and observed all storage conditions when working with this chemical3.

Hydrogel
The hydrogel, which is designed to encapsulate bacteria, is based on chitosan, beta glycerophosphate disodium salt, and hyaluronic acid. Hydrogels of this composition are safe for humans, biodegradable and biocompatible. This is confirmed by numerous studies and they are widely used in various drug delivery methods4.
Another thing that is important to add is that upon the degradation of hydrogel, bacteria number will be decreased by the immunomodulatory properties of the system. The idea is that an in situ injection of our system will cause an immune response because, as our tests have shown, the hydrogel tends to gradually degrade and release bacteria in small quantities, which in turn will attract various immune system cells and components to the injection site. In theory, they could have tumor-suppressive properties and help our system fight tumors as well

Colicin E1
Colicin E1 is a bacteriocin, a toxin native to E. coli that is released to fight other bacteria for nutrients or other resources5. The specificity and safety of the use of this toxin is ensured by the localization of delivery of the hydrogel itself and the system based on lactate, which is a marker of the tumor microenvironment

Preventing sepsis and inflammation
Many experts in the field of cancer and drug delivery, professors, and mentors advised us regarding the issue of sepsis because depending on the question of whether bacteria from our gel causes sepsis or not, we can determine the project's suitability for practical use6.
The cross-linking in our hydrogel and the pore size are on the scale of less than ten micrometers, which is quite enough for anti-cancer toxins to pass through these pores, but not for rapidly dividing bacteria. We experimented and tested different pore sizes each time until we achieved a size that could give us an accurate answer as to whether the hydrogel was safe, and whether the bacteria contained in it would not lead to unpleasant and deadly consequences such as bacteremia, septic shock, or inflammation
Also, it is important to say that the bacteria that are used in our project are DH5alpha and BL-21 and they are on the white list. Their release is not hazardous as was said above, they are encapsulated.

Kill-switch problem
In our original version of the plasmid, the quorum sensing and kill-switch mechanisms were present. The idea was that if the bacterium was released from the hydrogel and went to circulate through the blood vessels or other organs, it would not only stop producing the toxin because of Quorum Sensing by the AHL-induced LuxR gene but would also commit suicide so as not to cause sepsis6. Unfortunately, after consulting with experts in the field of immunology, we concluded that all the contents of the cell that will be released will, on the contrary, cause a stronger immune response and may worsen the patient's condition. Designed bacteria-loaded hydrogel systems will be slowly degrading in the tumor site while constantly releasing anti-cancer toxins. If bacteria will be growing and disrupting the inside of the gel, they will also release endotoxins which might cause unpredictable issues in situ7.
Therefore, the final version of our plasmid does not have such a mechanism. Also, it was found that overall it is not required for our system as the Hydrogel system will be injected in situ, which ensures the specific localization of the hydrogel and specific treatment.

Human Practices Safety


During the project, we conducted safety surveys among summer camp participants and NU community members regarding their knowledge of cancer. Besides, all the surveys were conducted inside our university anonymously. All the participants were properly informed in the polls and signed the consent form to avoid ethics rules violations.
An important aspect regarding the safety of the children who participated in our iGEM summer camp, since all of them were schoolchildren and many were under 18 years old, our team approached the issue of their safety with special attention. At the very moment when a summer camp participant crossed the gates of our university, members of our team met them and began to bear full responsibility for their safety. Regular headcounts, constant surveys regarding their needs, and constant supervision have ensured the highest possible safety that our team members could provide. More importantly, at the end of all activities associated with the summer camp, we made sure that all 41 students who chose the offline format were transferred to their parents or legal representatives. It is also important to note that the campus is a highly safe area, and the security office of Nazarbayev University allows entry only to those who are authorized to be on campus.

Future implementation


Our developed system and/or engineered bacteria are unlikely to leave laboratory conditions in the experimental stage. Even if the release occurs, bacteria are unable to survive in an external environment since they are auxotrophic and require specific conditions. Finally, when the project is fully developed as a drug it will be released beyond containment and injected into a patient’s organism but only after many years of clinical trials and FDA approval.
The design of our bacteria is built so that it specifically reacts to the microenvironment of a tumor rich in lactate, and for example, an injection of bacteria without hydrogel into a person will only cause an immune response and lead to the destruction of bacteria. Therefore, deliberate misuse is highly unlikely, only upon redesigning the bacterial genome.
Our system causes no side effects or any type of hazard to a human organism due to the multiple levels of safety that is imposed. Patients can even undergo simultaneous treatment with our system and any other therapy as no hazards of combined treatment are identified.
As our final will reach its potential users, the dosage of the drug can be calculated individually for a patient based on her tumor growth stage and overall treatment status. The treatment period with our system can be calculated according to the tumor size, its characteristics, and other factors. Meaning that bacterial concentration and respective drug release can be calculated specifically to a patient’s needs.

1. Trapotsis, A. (2023, September 26). Biosafety levels 1, 2, 3 & 4: What’s the difference? Consolidated Sterilizer Systems. https://consteril.com/biosafety-levels-difference/

2. Cornell University. (n.d.). Non-pathogenic escherichia coli strains biological agent reference sheet (bars). Non-pathogenic Escherichia coli Strains Biological Agent Reference Sheet (BARS) | Environment, Health and Safety. https://ehs.cornell.edu/research-safety/biosafety-biosecurity/biological-safety-manuals-and-other-documents/bars-other/non-pathogenic-escherichia-coli

3. Uc Santa Cruz. (2019). Ethidium bromide. https://ehs.ucsc.edu/lab-safety-manual/specialty-chemicals/ethidium-bromide.html

4. Ahmadi, F., Oveisi, Z., Samani, S. M., & Amoozgar, Z. (2015). Chitosan based hydrogels: characteristics and pharmaceutical applications. Research in Pharmaceutical Sciences, 10(1), 1–16. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/26430453

5. Schwartz, S. A., & Helinski, D. R. (1971). Purification and characterization of Colicin E1. Journal of Biological Chemistry, 246(20), 6318–6327. https://doi.org/10.1016/s0021-9258(18)61791-0

6. Van der Poll, T., & Opal, S. M. (2008). Host–pathogen interactions in sepsis. The Lancet infectious diseases, 8(1), 32-43. DOI: 10.1016/S1473- 3099(07)70265-7

7. Galanos, C., & Freudenberg, M. A. (1993). Bacterial endotoxins: biological properties and mechanisms of action. Mediators of inflammation, 2(7), S11–S16. https://doi.org/10.1155/S0962935193000687