Overview

Team Michigan continually prioritizes safety in all aspects of project design and lab work. We carefully selected reagents and parts that would minimize risk to our team members in the lab, to the surrounding spaces, to the environment, and to users of our final deliverable product. Additionally, we always follow the University of Michigan's and iGEM's safety standards, and hold each other accountable for being responsible and knowledgeable of lab practice.

Project Design

Much of our effort toward safety was made before entering the lab. We carefully designed our protocols to use reagents and parts that minimize risk wherever possible, which makes our research safer for ourselves, our environment, and anyone interested in using or investigating our project down the road.

Reagents

Since our project is oriented around DNA, we repeatedly run DNA gel electrophoresis. Many common gel electrophoresis stains such as Ethidium Bromide are known or suspected mutagens and are toxic to the environment. In order to evade the risks associated with such stains, we decided to use SYBR Safe stain in our gels, which has a similar sensitivity but is less hazardous than Ethidium Bromide and other stains1

In our qPCR experiments, we used SYBR Green I, a mutagenic chemical and standard tool for nonspecific dsDNA detection. In response, we took extra care to avoid contact with our skin, faces, and surrounding area by wearing proper PPE and exercising caution.

All of our reagents, including the aforementioned stains, are included on the iGEM whitelist and are also chosen to minimize risk to our members handling them. Inevitably, many of these reagents including buffers, acids and bases, detergents, and dyes are still at least minorly hazardous chemicals, often classified as skin irritants. Our lab space and safety training allow us to store and handle these chemicals quite safely.

Finally, we make sure that we use only non-pathogenic biological parts, including DNA and proteins, that are approved on the iGEM whitelist.

Human Cell Lines

Since our project involved testing human genomic DNA for the SNP located in the F2RL3 gene, it at first seemed like a natural choice to use blood to collect cells on which to perform our final assay. However, due to the added safety risks associated with handling human blood, we decided to turn toward immortalized cell lines instead. Specifically, we used the HEK293T cell line. This much safer source of human cells is approved on the white pages and was generously donated to our team via University researchers already utilizing them in their labs. It also provides future researchers who want to work with or build on our assay safer ways to do initial testing on human cells.

Living Organisms

For our project, the only other living organisms we used were BL21 E. coli, a well-established, non-pathogenic strain that is whitelist approved and safe for use in our Biosafety Level 1 lab. We always handled bacteria in an aseptic environment and wore fresh gloves, replacing them after each use. Moreover, we always disinfected surfaces before and after using 70% ethanol and made sure to dispose of any contaminated waste in our designated biohazard waste bin.

Fluorescence Reader

Part of our project involves a fluorescence reader, which we constructed ourselves, to monitor our diagnostic assay. We had a subgroup of our team involved in designing and building this part for our project. All of those members involved went through additional safety training relating to the specific machines and electronics used to construct the device.

The primary risk associated with the reader is the laser central to its operation. Due to its beams' ability to severely damage the eyes, our team members always wore special laser safety glasses while handling it. Moreover, the final construct was specially designed so that someone using it is shielded from the laser and does not need to wear any eye protection.

Lab Work

Our team used BSL1 and BSL2 lab spaces as appropriate for our work, all overseen by University of Michigan regulations.

Before starting any wet lab work, every member of our team completed University of Michigan laboratory safety training courses, including Chemical Laboratory Safety, Autoclave and Safety Procedures, and Bloodborne Pathogen Training for Research Labs. Additionally, the team underwent training with our advisors and appropriate University contacts and held personalized in-lab training with each member to firmly establish appropriate lab practice and safety techniques. This included learning proper disposal techniques in accordance with University standards, how to use important safety features such as the eyewash station, and what steps to take in case of an accident such as a chemical spill.

While working in the lab, our team members strictly adhere to proper laboratory attire (long pants, closed-toed shoes, etc.) and wear the proper personal protective equipment (PPE), including lab coats, goggles/eye protection, and gloves. Many of our reagents, such as stains (mentioned above in project design), buffers, antibiotics, etc., are classified as irritants and biohazards, so our PPE and chemical safety practices help protect us and outside areas from these substances. Specifically, we always wear a properly fitting lab coat with gloves up to the cuffs and eye protection to minimize the risk of chemicals coming into contact with skin. We make sure to disinfect the benches with 70% ethanol before and after use, and we work under a fume hood when handling volatile chemicals. When using microbial agents, we always operate in a sterile environment inside the hood and dispose of biologically contaminated waste in the biohazard waste bucket in accordance with University standards. Moreover, we autoclave our glassware and other containers to completely sterilize the materials before and after use, which both protects our experiments from contamination and protects us from foreign biological agents.

Waste Disposal

We made sure that our waste materials were disposed of in their respective bins. Pipette tips, gels, Eppendorf, and Falcon tubes that were used during experiments were disposed of in biohazard waste bins, the contents of which were subsequently autoclaved and disposed of by experienced personnel. To dispose of chemical products, we placed all liquid wastes in a specially designated chemical waste container and solid wastes in the biohazard waste bucket. Additionally, any sharp objects needing to be disposed of were properly separated out into sharps disposal. This ensured the safety of lab and facility personnel by minimizing the risk of unexpectedly coming into contact with sharp objects. Our waste disposal methods prioritize the health of lab and community members, as well as the health of our local environment.

Compliance

During the course of our research, we kept in close contact with our Classroom Services Supervisor, Margo Gebbie, Ph.D., to ensure that we were upholding lab safety and respecting University of Michigan lab research standards. She and our team leaders made sure that all members checked in and out of the lab when they did research. This allowed us to keep track of who was working and when, and could help us keep our teammates accountable in the event that the lab was not cleaned up properly or if there were to be an accident.

Future Project Applications

We intend for our final diagnostic kit to be easily and safely operated in any clinical setting with access to electricity. In order to ensure safe handling, the kit would include step-by-step instructions that could be easily followed by any individual with a basic level of medical or lab training. These instructions would place particular emphasis on handling human specimens safely and with careful sterile technique to yield a safe, easy, and accurate result.

SNP Detection Kit Instruction include sample collection, cell lysis, DNA extraction, LAMP, and fluorescence detection.