Left Hex

Safety

Right Hex

Overview

Team Michigan consistently prioritizes safety in project design and lab work. We carefully selected reagents and components to minimize risks to our team members, the surrounding areas, the environment, and the users of our bioreactor. Furthermore, we consistently followed the University of Michigan's and iGEM's safety standards and held each other accountable for being responsible in the lab. Some of the primary ways we prioritized safety include the following:

  • Using non-pathogenic E. coli and P. putida to safely and effectively express genes via plasmid expression vectors
  • Replacing mutagenic ethidium bromide with SYBR Green
  • Implementing proper toxic and hazardous waste disposal protocols
  • Learning how to contact Environmental, Health, & Safety (EHS) and report medical incidents at our academic institution
  • Learning how to operate GC-FID and GC-MS machines safely


Project Design

To prioritize safety, our team made substantial efforts before entering the lab. We consulted institutional safety managers to assess the risks associated with our materials and protocols. Key considerations included selecting non-pathogenic BSL-1 strains of DH5α E. coli and S16 P. putida for our experiments, which negated the need for medical surveillance for bacterial infections. We also chose safer nucleic acid stains and incorporated feedback to refine our approach. Additionally, we implemented meticulous disposal and cleaning protocols to minimize environmental contamination by gentamicin-resistant bacteria.

Reagents

Our project focused on plasmid assembly and insertion, necessitating frequent runs of DNA gel electrophoresis. Traditional gel stains like Ethidium Bromide are known mutagens and pose environmental risks. To mitigate these dangers, we opted to use SYBR Green stain in our gels. SYBR Green offers similar sensitivity to Ethidium Bromide but significantly lower toxicity and environmental impact.

All of our reagents, including the SYBR Green stain, are on the iGEM White List and were selected to minimize risk to our team members. While many of these reagents, such as buffers, acids and bases, detergents, and dyes, are minorly hazardous and classified as skin irritants, our lab space and safety training enabled us to store and handle these chemicals safely.

Since our project focuses on the degradation of 1,4-dioxane, a Group 2B carcinogen, we have implemented strict precautions to ensure safe handling in our lab. Due to the volatile nature of pure 1,4-dioxane, we always worked in a fume hood at ANSI/AIHA Z9.5-2012 standards for laboratory ventilation (suitable for dioxane handling), with the sash at the proper level, to minimize exposure to 1,4-dioxane vapors1. Furthermore, we created 100x diluted stocks of 1,4-dioxane in water, significantly reducing its volatility. Any culturing work involving 1,4-dioxane was performed with the diluted 1,4-dioxane. Additionally, the 1,4-dioxane purchased was stabilized with 30 ppm BHT, which prevents harmful reactions2. Timepoint samples from degradation testing were frozen at -80°C, at which point 1,4-dioxane freezes, including when mixed with water. The pure stock ordered was stored in an airtight container in a cool, dark, ventilated environment in a School of Public Health lab certified to handle DX, PFAS, and similar chemicals.

Regarding the organic materials utilized in our lab work, the growth media used for our project (Luria Broth, LB) does not contain nutrients or compounds that will adversely react with 1,4-dioxane3. Finally, we made sure to use only non-pathogenic biological parts, including DNA and proteins, that are approved on the iGEM White List.

Living Organisms

For our project, we used DH5α E. coli and S16 P. putida, which are well-established, non-pathogenic strains that are White List 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 disposed of any contaminated waste in our designated biohazard bin. Proper disposal of biohazardous waste (via autoclave) was performed by experienced personnel.

Machinery and Unfamiliar Techniques

To measure the 1,4-dioxane concentrations, we used gas chromatography (GC) with flame ionization detection (FID) to detect the carcinogen's presence. We worked with the Manz Lab here at the University to learn how to use the machine. Specifically, we learned how to calibrate it to create a standard curve of decreasing dioxane concentrations from a standard. This was all done under the supervision of Dr. Manz and Rachel Klein, a PhD student in the Manz lab, to ensure all safety protocols were followed without fail.

We also used GC with mass spectrometry (MS) to confirm our results further. We worked with the Pennathur lab to learn how mass spectrometry provides an analytical finish. The internal dioxane standards were stored at 4°C while the authentic dioxane standards were stored at room temperature in a flammable compounds cabinet in the Pennathur lab, per established storage guidelines. Both standards and their respective dilutions were prepared in a fume hood to prevent any dioxane that may evaporate from spreading through the lab space. Vials made for the machine were securely crimped and moved between the hood and GC machine as quickly as possible. Dr. Subramaniam Pennathur, Dr. Jaeman Byun, Dr. Manikanta Arnipalli, and Dr. Saroj Chakraborty supervised the work to strictly adhere to all safety protocols.


Lab Work

Safety

Safety in the lab was the top priority for our team to protect ourselves, others, and the validity of our results. Before starting wet lab work, every team member 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, appropriate University contacts, and personalized in-lab training with each member to firmly establish appropriate lab practice and safety techniques.

University safety managers assisted us in designing essential safety measures required before commencing lab work. They instructed us on properly disposing of hazardous chemicals, biohazard waste, and sharps and informed us about the locations of emergency showers, eyewash stations, and spill kits. At the beginning of our project season, we explained these risks and protocols to all members in a meeting to ensure the safety of everyone involved throughout our experimentation timeline. Integrating safety measures into our experimentation and developing safety plans to protect all students and others was vital to our project's design. We only began conducting our project under the condition that all members were fully aware of safety protocols both in and outside the lab.

A clear communication plan was established for lab students, detailing contacts for minor or significant emergencies, including phone numbers for the building’s lab manager and the University of Michigan’s environmental health and safety department. Our lab manager, Dr. Margo Gebbie, and Undergraduate Science Building (USB) employees were always available to answer questions or provide support in the lab space. In cases where lone work was necessary, the student organization’s board members and advisors were reachable via text, call, or Slack message, though their assistance was rarely needed. USB building employees and our organization’s advisor were designated incident-reporting contacts. They would have helped us complete any necessary reports in the event of an incident.

Protocol & Protective Equipment

While working in the lab, our team members strictly adhered to proper laboratory attire (long pants, closed-toed shoes, etc.) and wore personal protective equipment (PPE), including lab coats, goggles/eye protection, and gloves. Students were required to wear PPE at all times in the lab, even if experimentation was not actively occurring. Many of our reagents, such as stains, buffers, and antibiotics, 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 disinfect the benches with 70% ethanol before and after use and work under a fume hood when handling volatile chemicals. When working with standards, we prepared them in a fume hood to prevent any 1,4-dioxane that may evaporate from spreading through the lab space. Vials made for the machine were securely crimped and moved between the hood and GC machine as quickly as possible. While 1,4-dioxane does not absorb through skin, as the primary mode of uptake is through drinking/ingestion, we took all proper precautions through PPE and handling to minimize exposure to the chemical4.

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 per university standards. Moreover, we autoclave our glassware and other containers to completely sterilize the materials before and after use, which protects us and our experiments from contamination and foreign biological agents.

Because we work in a shared lab space with university classes and other labs, we are incredibly diligent about safely disposing of hazards and correctly using the equipment. Topics learned in safety and security training included the following:

  • Lab access and rules (e.g. appropriate clothing, eating and drinking)
  • Responsible individuals (e.g. lab or departmental specialist or institutional biosafety officer)
  • Differences between biosafety levels
  • Biosafety equipment (e.g. biosafety cabinets)
  • Proper microbial technique
  • Disinfection and sterilization
  • Emergency procedures
  • Rules for transporting samples between labs or shipping between institutions
  • Dual-use research and/or experiments of concern
  • Chemical, fire, and electrical safety

We communicate frequently with Dr. Margo Gebbie, the Classroom Services Supervisor, to ensure we are upholding the lab safety standards designated by the University of Michigan.

We implemented multiple measures to control our inventory, access to physical materials, and access to our online data. For inventory, we had a designated cabinet and refrigerator in our laboratory space to properly store chemicals and reagents. All materials were labeled thoroughly with the name of the substance inside, the date obtained, the person to open or make the reagent, and our club’s acronym designating that it belonged to our group; this prevented students in the laboratory space or building employees from harming themselves with our materials. We kept our materials in a locked room, allowing access only to those with the entrance code or granted permission. For data, we collected and stored information in Benchling, Slack, and Google Drive. These sites require passwords or special permissions, which we grant only to our members and advisors.

Waste Disposal

We ensured the proper disposal of our waste materials by placing them in their respective bins. We disposed of pipette tips, gels, Eppendorf, and Falcon tubes used during experiments in biohazard waste bins. Experienced personnel autoclaved and handled the contents. We placed liquid chemical wastes in specially designated chemical waste containers and solid chemical wastes in the biohazard waste bucket. We separated sharp objects into sharps disposal containers to prevent injuries and minimize the risk of accidental contact. We separated sharp objects into sharps disposal containers to prevent injuries, minimizing the risk of unexpected contact. These waste disposal methods prioritized the health and safety of lab and community members and our local environment.

For waste containing 1,4-dioxane, we meticulously separated all solid and liquid waste from other biohazardous materials to ensure proper disposal. We bleached liquid cultures containing 1,4-dioxane and then disposed of them in glass jugs, adhering to University of Michigan EHS guidelines. We added all other liquids, including 1,4-dioxane waste, directly to these labeled jugs. EHS personnel collected the disposal jugs directly from our laboratory, eliminating the need for lab members to transport them. For solid waste contaminated with 1,4-dioxane, we allowed the compound to evaporate entirely in the hood before disposing of the waste through EHS.

A significant risk in our lab space is working with gentamicin-resistant bacteria, which can contaminate water sources with live antibiotic-resistant bacteria. To ensure proper disposal, we bleached contaminated glassware and disposed of materials in the biohazard waste bins provided by the University. We followed all disposal and cleaning instructions closely to ensure the safety of our students and collaborators.

Compliance

During our research, we kept close contact with our Classroom Services Supervisor, Margo Gebbie, Ph.D., to ensure that we upheld lab safety and respected University of Michigan lab research standards. She and our team leaders ensured that all members checked in and out of the lab when researching. This system allowed us to track who was working and when, and it helped us hold teammates accountable if someone did not clean up the lab correctly or if an accident happened. The Manz and Pennathur labs supervised GC-FID and GC-MS testing, respectively. All safety regulations, including proper liquid and solid waste disposal standards, were upheld.


Future Applications

Ideally, we will apply our project to a bioreactor that we will implement in a water treatment facility. We will grow P. putida S16 in a biofilm placed on disks within the rotating bioreactor to ensure proper aeration. During our proof-of-concept testing, we will conduct bioreactor testing in a hood using an open-topped reactor, allowing trace amounts of 1,4-dioxane to minimize volatilization from the water. Our bioreactor will use disks to facilitate biofilm formation, and we will wear proper PPE when transferring the disks within the hood. We will bleach and dry any used disks and always contain them within the hood to protect the lab space from any residual 1,4-dioxane on the disks and allow their reuse.

To further ensure bacterial removal, we will use chloramines, organic matter filters, and UV treatment in the water treatment facility to prevent the spread of genetically modified bacteria. Additionally, we will employ chemical scrubs to remove dioxane in tandem with biodegradation. We will place the bioreactor in the middle of the current treatment process to ensure the following steps can clean residual products from our system.

Calculations

Calculations for Evaporation of 1,4-Dioxane from Aqueous Media:

Given:

  • Concentration of 1,4-dioxane: 0.1174 M (at maximum)
    • Note that this is if the entire 20 mL 1:100 stock was left open to evaporate - in reality, the amount used was significantly less, as 48.35 uL was placed in 5 mL of LB.
  • Volume of water: 20 mL
  • Henry's Law constant: kH = 4.8 x 10-6 atm·m3/mol
  • VFumehood = 1000 L

Steps:

  1. Calculate the number of moles of 1,4-dioxane (denoted DX) in the solution:

    MolesDX = 0.1174 M * 0.02 L = 0.002348 mol

  2. Use Henry's Law to find the equilibrium partial pressure of 1,4-dioxane in the air:

    PDX = kH * MolesDX = 5.64 * 10-7 atm

  3. Find the concentration of 1,4-dioxane in the air at equilibrium:

    Cair = n/V = P/RT = (5.64 * 10-7 atm)/((0.0821 L*atm/mol*K) * 98 K) = 2.37 * 10-8 M.

  4. Calculate the total amount of 1,4-dioxane in the air space above the solution:

    Assuming 1000 L is the volume of the fume hood, we have:

    MolesDXinAir = Cair * VFumeHood = 2.37 * 10-5 M.

  5. Compare the moles of 1,4-dioxane in the air to the initial moles in the solution:

    Fraction Evaporated = (2.27 * 10-5)/0.002348 = 1.01 * 10-2 = 1.01%

References