Laboratory
From wet lab to hardware, safety is an integral part of our experimentation. As such, this page details the means by which Lambert iGEM created a safe lab environment. We cover general safety procedures all members follow, as well as specific procedures each subcommittee follows.
Team Safety
- Wash hands coming in and out of the laboratory.
- Protect eyes, mucous membranes, open cuts, and wounds from contact with biohazard material.
- Do not eat or drink when in the lab area.
- Always use gloves and splash-proof goggles.
- Tie back loose or long hair.
- Disinfect all surfaces with 70% ethanol before working.
- Disinfect all disposable tips, glassware, and tubes by soaking in 10% bleach solution for 20 minutes and then disposing in normal waste.
- Dispose of growth plates by disposing them into a biohazard container which gets autoclaved.
- Check all equipment for good working order: no chips, torn chords, or cracks. Report any issues to an instructor immediately.
- When pipetting, do not touch the tip to the side of the container.
- Do not lay caps of tubes upside down. Use masking tape to hold the bottom of the cabinet.
- Clean work area with 70% ethanol after working.
- Clean up all glassware and labware before leaving the lab.
- Place all backpacks and stools to the side of the lab to keep walkways clear.
- Always know the correct procedure for the disposal of lab materials.
Project Safety
Overview
Our device, CADlock, used more accessible point-of-care testing to detect upregulated microRNA related to coronary artery disease (CAD). Specifically, our project targeted hsa-miR-1-3p and hsa-miR-133a-3p. To ensure the proper success of our design, we also utilized hsa-miR-451-3p as a control throughout our experiment. CADlock will be used conjointly with current medical diagnostic testing and for further research into the effects of miRNA on CAD. The team used a padlock probe sensing mechanism coupled with rolling circle amplification (RCA) and rolling circle transcription (RCT) to produce a quantifiable fluorescent output. RCA utilizes linear DNA probes (BBa_K4245130 and BBa_K4245132) and the Lettuce aptamer (BBa_K4245133), and RCT works through the Broccoli aptamer (BBa_K3380153). However, our team attempted to characterize similarly-behaving Spinach (BBa_K734002), iSpinach (BBa_K3380150), and iSpinach-D5-G30-A32 (BBa_K4245000), which was the initial reporting mechanism of RCT. In addition, we also characterized linear DNA probes.
Wetlab
| E. coli Strain | Purpose | Pathogenicity | Health/ Environmental hazards | BL21 | Transformation | Nonpathogenic-for lab cloning | Extremely limited-Biosaftey Level 1 Lab | DH5-alpha | Transformation | Nonpathogenic-for lab cloning | Extremely limited-Biosaftey Level 1 Lab | NEB 10-beta | Transformation | Nonpathogenic-for lab cloning | Extremely limited-Biosaftey Level 1 Lab |
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A solution of 1% sodium hypochlorite and 70% ethanol will kill biosensor cells. Results show that a 1% sodium hypochlorite solution sprayed on the surface and let sit for 10 minutes will effectively remove all DNA, saliva, blood, semen, and skin cells from any smooth or pitted surface when wiped down with 70% ethanol afterward. However, sodium hypochlorite solution followed by ethanol can produce amounts of gaseous chlorine above recommended exposure levels. As a result, 1% sodium hypochlorite followed by distilled water was tested and proven to be effective as well (Kaye et al., 2015).
Hardware/Software
We prototyped Micro-Q using 3D printers, which pose potential safety risks because certain parts, like the plastic-melting nozzle, can heat up to 220°C. Furthermore, leaving the 3D printer unattended would be dangerous because its high temperatures could cause a fire.
Precautions:
- Crash detection and heat overload software built into the printers
- Team member or PI is present during printing to intervene in case of an accident
Micro-Q’s outer box and structure are made out of laser-cut pieces of wood. Laser cutters produce lasers strong enough to cut wood but also strong enough to overheat and cause a fire.
Precautions:
- No flammable materials exposed to laser
- Encapsulated in a plastic case with UV protection
- 2 ft tape around the laser cutter where students are not allowed to be inside without teacher supervision while the machine is running
- UV glasses are required when working with Micro-Q or the laser cutter
Micro-Q was built and prototyped following proper safety precautions while working with the electronic devices. Whenever working with electronics such as the Arduino or an ESP-32 microcontroller, the devices are placed on antistatic materials. When using power supplies or AC adapters, we take precautions such as checking the devices for damage before use and making sure all liquids, food items, and potentially flammable materials are kept away. While soldering electronics, team members wear safety glasses, use electrically and thermally insulated gloves, and work in a well-ventilated and isolated environment.
Human Practices and Education
Participants in all surveys provided signed consent for the release of their responses from themselves or a legal guardian. Additionally, participants in all events hosted by Lambert iGEM provided consent for photo and video release. Proper safety instructions and procedures were given during the in-person and virtual camp activities.
Implementation
As we work in a Biosafety Level 1 (BSL-1) laboratory, Lambert iGEM was unable to extract miRNAs from and test our reactions in human blood. However, we received human pooled serum for our experimentations from a local university, which contains no blood pathogens and/or bioengineered products. While testing with serum, we wore personal protective equipment (PPE) including nitrile gloves, splash goggles/safety glasses, and lab coats to prevent body contact with reagents as well as contamination of samples. Regarding disposal, we autoclaved all treated serum samples and carefully discarded them down the lab sink while wearing PPE to prevent splash-back. Afterward, we followed the general hand washing protocols.