Safety

Keeping our team safe

    CONTENTS

Our Project

Our project builds upon our 2022 work in which we designed an E. coli-based GFP biosensor for the detection of manganese contamination in water and demonstrated a detection limit down to 0.01mM manganese chloride, or about 0.55 mg of manganese per liter of water. This lands just above the health advisory level of 0.3 mg of manganese per liter of water. Our 2023 team is building upon this work by adapting our sensor to a cell-free platform to improve sensitivity and signal from our fluorescent reporter. We have done some initial testing with a home-made cell-free lysate and observed GFP production by our sensor in response to 0.1mM MnCl2, thus demonstrating feasibility of our approach. Our cell-free method utilizes standard protocols and general lab equipment. We hope to continue optimization of this sensor in cell-free experiments with the goal of developing a fieldable test strip using freeze-dried cell lysate. We are also working on a strategy to chelate manganese from drinking water for use in conjunction with our manganese biosensor. We are planning to test several heavy metal binding proteins to identify a protein capable of sequestering manganese from drinking water samples. This approach could be used to generate portable water purification filters that could be used in remote areas.

Our Lab

Here is the main bacterial culture bench space with 70% ethanol for wiping down the benchtop before and after bacterial work, a biohazardous waste container for waste dry goods, a sharps waste container (if needed) and kimwipes.

Next is our main work area with disposable gloves, sharps disposal, lab coat, safety goggles, 70% Ethanol for wiping down the benchtop before and after experiments, and gloves for hot and cold sample handling. Bleach (not shown) for treating liquid culture waste is kept under the lab sink.

Our Experiments

  1. Our 2023 team has validated the BBa_K4217003 manganese biosensor for use in cell-free experiments. Originally validated for use in E.coli culture-based testing, the team tested the sensor plasmid in cell-free assays and demonstrated its functionality. Optimization of the sensor plasmid for use in cell-free assays involve testing of different promoters (e.g. T7) and reporter proteins (e.g. luciferase) to improve our sensor performance.
  2. Our planned efforts to synthesize various affinity-tagged manganese-binding proteins to identify peptides capable of binding manganese were delayed and implementation of our sequestration strategy may occur in the future.

Our Chemicals

  • Ethidium Bromide, a mutagen and potential carcinogen, may be used in DNA gels during plasmid preparation if SYBR green staining is insufficient. Appropriate PPE and disposal guidelines are used.
  • Manganese, a heavy metal, is used in this project. MnCl2 is used as a manganese source to test the effectiveness of our sensor. MnCl2 treated samples are disposed of with the assistance of the Wright State Environmental Health and Safety Department in accordance with state and federal guidelines.

Possible Risks

While there are some possible hazards that can come with the work that we're doing, we make sure to take necessary precautions to make sure that all risks are minimized. We use standard E.coli MG1655 and BL21(DE3) strains which require BSL-1 standard precautions. Appropriate PPE and disposal guidelines are used when working with ethidium bromide. Basic safety precautions including gloves, lab coats, and eye protection are used to minimize risk when working with manganese chloride.

Risk Management

Here are some of the ways that we work to manage the risks that come with the work in our lab:

  • Our laboratory utilizes CITI safety training; the following modules cover our work: Basic Introduction to Biosafety, Conflicts of Interest, Hazard Communication, Initial Biosafety Training, Lab Chemical Safety, and OSHA Bloodborne Pathogens. This extensive training is designed to provide team members with safety training relevant to their project and additional training relevant to general lab research more broadly. All training was completed before students worked in the lab (in Apr - May).
  • We have contacts at Air Force Research Laboratories at Wright Patterson Air Force Base that work with bacterial-based sensors of heavy metal contamination and colleagues at water monitoring facilities that we will consult about the potential risks associated with a manganese biosensor of water contamination.
  • Biosafety regulations are put forth by the HHS and USDA Select Agent Regulations (42 CFR part 73, 9 CFR part 121, 7 CFR part 331) and enforced at our institution by the Wright State Environmental Health and Safety department (https://www.wright.edu/environmental-health-and-safety).
  • We have our faculty mentors available to consult with any concerns we may have regarding safety and risk management. In addition, our CITI training has given us the knowledge base required to be safe and successful in our studies. We have various methods of cleanup in the laboratory available (e.g. 10% bleach, 70% ethanol) at every bench that are used to disinfect both our workspace and our waste products. We also wear gloves, safety goggles, and lab coats to keep ourselves safe.