Safety

A Comprehensive Guide on SHIELDing Ourselves

Safety

Laboratory

Safety is a fundamental aspect of our experimentation, spanning from the wet lab to hardware applications. This page serves as a comprehensive guide outlining the safety measures implemented by Lambert iGEM to establish a secure laboratory environment.

Team Safety

  • Wash your hands when entering and leaving the lab.
  • Protect your eyes, mucous membranes, open cuts, and wounds from biohazard materials.
  • Do not eat or drink in the lab.
  • Always wear gloves and splash-proof goggles.
  • Keep long or loose hair tied back.
  • Before starting work, disinfect all surfaces with 70% ethanol.
  • Soak disposable tips, glassware, and tubes in a 10% bleach solution for 20 minutes before disposing of them in regular waste.
  • Dispose of growth plates in a biohazard container that will be autoclaved.
  • Check that all equipment is in good condition, with no chips, torn cords, or cracks.
  • Report any problems to an instructor immediately.
  • When pipetting, avoid touching the tip to the side of the container.
  • Don’t place tube caps upside down; use masking tape to secure the bottom of the cabinet.
  • After working, clean your area with 70% ethanol.
  • Ensure all glassware and lab equipment are cleaned before leaving.
  • Keep walkways clear by placing backpacks and stools to the side.
  • Always be aware of the proper disposal procedures for lab materials.

Project Safety

Overview

Antimicrobial resistance presents significant challenges due to the increasing costs and vulnerabilities of current treatment approaches. In response, Lambert iGEM 2024 created SHIELD: a versatile toolbox for developing novel antimicrobials that combat antibiotic-resistant diseases. Our project was focused on a CRISPRi system to downregulate the critical genes in antibiotic-resistant bacteria, paired with toehold biosensors to measure the effectiveness of gene silencing. SHIELD enables the targeting of multiple genes which were used in developing customized antimicrobials for each disease.

Wetlab:

E.Coli StrainPurposePathogenicityHealth/Environmental hazards
BL21TransformationNonpathogenic-for lab cloningExtremely limited-Biosafety Level 1 Lab
DH5-alphaTransformationNonpathogenic-for lab cloningExtremely limited-Biosafety Level 1 Lab

CRISPRi:

In all experimentation for CRISPRi, safety protocols were adhered to. Plated plasmids for dCas9 and GFP were provided by Dr. Vincent Noireaux. It must be noted that the tuberculosis bacteria itself or genes directly from the bacteria were never used at any point of experimentation. All DNA/RNA and isolated gene fragments were ordered as linear constructs using IDT. The CRISPRi system was tested in commercial cell-free TXTL systems instead of bacterial cells, avoiding the use of live cells. Additionally, our target gene for tuberculosis, inhA, is unique to the tuberculosis bacteria, and is not found in humans.

Discarding cells in the field

Biosensor cells can be killed with a solution containing 70% ethanol and 1% sodium hypochlorite. The findings indicate that any smooth or pitted surface can be successfully cleaned of all DNA, saliva, blood, semen, and skin cells by spraying it with a 1% sodium hypochlorite solution, letting it sit for 10 minutes, and then wiping it down with 70% ethanol. However, amounts of gaseous chlorine above the recommended exposure levels can be produced by ethanol followed by sodium hypochlorite solution. Thus, it was determined through testing that 1% sodium hypochlorite followed by distilled water was also effective (Kaye et al., 2015).

Toeholds:

The disease we used as our proof of concept, Tuberculosis, is a very dangerous pathogen to work with and is of a Biosafety Level that our high school does not have access to (Herman et al. 2006). Therefore, we decided to use DNA fragments of the Tuberculosis genes that we want to target, purchased from IDT and Twist, to eliminate exposure or infection. The use of DNA fragments required the need to work in cell free, for which we utilized the myTXTL Pro Kit to perform our toehold experiments in.

Cell Free:

In the Cell-Free extraction process, proper PPE such as lab coats, gloves and safety goggles were used to prevent contamination. Competent BL21 cells were used in the cell growth and extraction process. BL21 cells are a strain of E.coli, commonly used in biotechnology and classified as Biosafety Level 1. The equipment used to handle the BL21 cells were properly cleaned with bleach after use to destroy any residual biological materials.

HP-Wetlab:

In our soil testing, we did not use any DNA from cultures of living organisms. Lysis solution was used in the DNA extraction process to ensure that all DNA is not alive. Proper PPE was used in soil extraction, PCR, and gel electrophoresis. Additionally, all locations consented to giving us access to collect soil samples from their facilities.

Software:

In developing SWORD, a machine-learning tool for designing toeholds, we used data from a verified research source that was cleaned to remove bias. Before testing any designed sequences in experimentation, we verified any toeholds designed with NUPACK, the existing state-of-the-art for designing toeholds.

Electronic Devices:

LabPilot was designed and prototyped with strict adherence to safety protocols when handling electronic components. For example, Arduino microcontrollers and similar devices are always placed on antistatic surfaces during use. When utilizing power supplies or AC adapters, we ensure the devices are inspected for any damage beforehand and keep liquids, food, and flammable materials at a safe distance. During soldering tasks, team members wear safety goggles, use insulated gloves for both heat and electricity, and work in a well-ventilated, isolated space. Eye protection and proper thermal management were also prioritized when using drills. After completing work on any electronic device, we ensure it is completely powered down and disconnected, including removing batteries when applicable.

3D Printer:

We developed LabPilot prototypes using 3D printers, which can present safety hazards due to components like the plastic-melting nozzle, capable of reaching temperatures up to 220°C. Additionally, leaving the 3D printer unattended poses a fire risk because of these high temperatures. Precautionary Measures: Built-in crash detection and heat overload software in the printers A team member or the PI is always present during printing to respond in case of an emergency.

Drill:

To assemble the components of LabPilot, the Lambert iGEM team utilized a drill, which poses risks such as getting entangled with personal items and causing injury. Precautionary Measures: Wore personal protective equipment (PPE), including gloves and safety glasses Powered off the drill when not in use Secured all loose belongings to prevent entanglement

Human Practices and Education:

Participants from all surveys provided signed consent for the release of their responses from either themselves or their guardians. Additionally, participants or their guardians provided consent for photo and video footage. Proper safety instructions and procedures were given.

References:

Herman, P., Fauville-Dufaux, M., Breyer, D., Van Vaerenbergh, B., Pauwels, K., Dai, C., Thi, D., Sneyers, M., Wanlin, M., Snacken, R., & Moens, W. (2006). Biosafety Recommendations for the Contained Use of Mycobacterium tuberculosis Complex Isolates in Industrialized Countries. https://www.biosafety.be/sites/default/files/mtub_final_dl.pdf ‌