Safety

Introduction

The 2023 Technion IGEM team prioritizes safety, ensuring the well-being of our students, our users and the environment. All team members have undergone comprehensive safety training for both general laboratory work and microbiological procedures related to our project before commencing wet lab activities.

Lab procedures

Our laboratory works strictly adhered to approved protocols and received mentor and host approvals, in coordination with the Technion's safety unit. All project materials used were from the IGEM white list. We were trained in lab safety practices, including the use of safety showers, eye wash stations, and fire extinguishers. We were also familiarized with fire alarms, emergency numbers, and gas valve locations. Throughout the project, lab members worked in pairs, and every protocol was preceded by a briefing. A shadow procedure was implemented for late-hour protocol execution, with a designated person who was responsible for overseeing lab access.

Our work included Biosafety Level 1 organisms: E. coli TOP 10 [1], E. coli DH5α [1], Bacillus subtilis 168 [2], and Lactobacillus crispatus [3]. We took protective measures by wearing gloves, closed-toe shoes, long pants, and lab coats. Our benches were regularly sanitized before and after each protocol.

Waste disposal

We have separated between three types of waste.

• Biological waste (bacteria related). This waste was autoclaved before disposal.
• Chemical waste (organic solvents related). This waste was put in chemical hoods and disposed of by Technion's authorized personnel.
• General waste (no relation to the other two types of waste).

Protect

In the pursuit of integrating our system with the genome of L. crispatus for sustained efficacy, as elaborated in the description of our project, we recognized the importance of incorporating a fail-safe mechanism, a "killswitch", to enable the controlled elimination of our engineered bacteria in case of unforeseen issues, thus ensuring user safety.

Our design aims to incorporate a repressor protein with a constitutive promoter and the selected toxin with an inducible promoter into the bacterial genome. Initially, we developed the killswitch for B. subtilis as a model and later adapted it for use in L. crispatus.

Compete

Our initial concept was to equip L. crispatus with FimH, the same adhesin used by UPEC, to manifest competition and also promote better adhesion of L. crispatus to gain a microbiota advantage.

However, we quickly abandoned this idea, recognizing FimH as a potent pathogenicity factor [4]. Studies indicated that FimH was crucial for UPEC invasion, with FimH-deficient UPEC being unable to invade cells, while beads coated with FimH were internalized [4]. This arises from FimH's ability to bind to various receptors, including Integrins, integral membrane proteins that normally link the extracellular matrix to the actin cytoskeleton [4]. Considering these findings, we understood the potential risk of inducing pathogenicity by expressing FimH in L. crispatus. Putting safety and ethics as main priorities in our project, we decided to not pursue this idea further.

Ecological Component

The uncontrolled release of genetically modified organisms (GMOs) into the ecosystem or sewage systems poses significant environmental risks in theory. Unchecked GMO proliferation could theoretically disrupt microbial communities, impacting plants, animals, and human reliance on these ecosystems. Additionally, the theoretical spread of GMOs could lead to unintended genetic interactions, creating unpredictable hybrids with potential harm to native species and higher trophic levels.

To theoretically address these concerns, we propose implementing a second killswitch system designed for release in sewers and the environment. This system would allow for the rapid termination of GMOs in the event of a theoretical outbreak, preventing unintended environmental spread, and safeguarding ecological balance while maintaining theoretical control. This theoretical, proactive measure is vital for the responsible and safe use of GMOs in our endeavors. You can read more about it on ours future plans page.