Objetives

Our objectives in this section are to cover the following:

To achieve all our proposed goals, a "biosafety workshop" was conducted where the entire iGEM Bolivia team was informed about the risks and how to minimize them, from entering the laboratory to managing potential spills and their disposal.
When performing our objectives 1 and 2, strict biosafety protocols are essential to ensure safety and prevent contamination or accidental release of genetically modified organisms (GMOs). Here is a list of the practices and a summary of what was covered in the workshop

Laboratory Classification and Access Control

• Biosafety Level (BSL): Ensure that the laboratory is classified at the appropriate biosafety level (typically BSL-1 or BSL-2 for most bacterial transformations) depending on the risk assessment of the organisms used.
• Access Control: Restrict access to the laboratory to authorized personnel only, and ensure that all individuals entering the lab are trained in the relevant biosafety procedures.

Personal Protective Equipment (PPE)

• Protective Clothing: Wear lab coats, gloves, and safety goggles at all times to prevent exposure to hazardous materials.
• Hand Hygiene: Wash hands before and after handling bacterial cultures, as well as after removing gloves.

Containment Practices

• Use of Biological Safety Cabinets (BSCs): Perform bacterial transformations within a Class II Biological Safety Cabinet to contain aerosols and prevent the release of genetically modified organisms.
• Closed Systems: Utilize closed containers and systems whenever possible to limit exposure and prevent spills or leaks.

Disinfection and Sterilization

• Surface Disinfection: Regularly disinfect work surfaces before and after the transformation process with appropriate disinfectants such as 70% ethanol or a bleach solution.
• Sterilization of Equipment: Autoclave all reusable equipment, such as pipettes and culture tubes, before and after use.

Waste Disposal

• Biological Waste: Dispose of bacterial cultures, transformation reagents, and contaminated materials in designated biohazard bags and autoclave them before final disposal.
• Liquid Waste: Treat liquid waste, including culture media and wash solutions, with disinfectants before disposal.

Accident and Spill Response

• Spill Mercury Containment Kits: Have a spill kit on hand with absorbent materials, disinfectants, and personal protective equipment to manage accidental spills.
• Incident Reporting: Immediately report and document any spills, exposures, or accidents to the laboratory supervisor or biosafety officer.

Training and Competency

• Staff Training: Ensure all personnel are trained in biosafety protocols, bacterial handling, and emergency procedures.
• Ongoing Competency: Regularly update training and review safety procedures to maintain high standards of biosafety.

Documentation and Record Keeping

• Logbooks: Keep detailed records of all bacterial transformation activities, including the organisms used, procedures followed, and waste disposal methods.
• Biosafety Audits: Every laboratory where we made our work, conducts regular audits of laboratory practices and biosafety protocols to ensure compliance with established guidelines

Disinfection and Sterilization

• Surface Disinfection: Regularly disinfect work surfaces before and after the transformation process with appropriate disinfectants such as 70% ethanol or a bleach solution.
• Sterilization of Equipment: Autoclave all reusable equipment, such as pipettes and culture tubes, before and after use.

Waste Disposal

• Biological Waste: Dispose of bacterial cultures, transformation reagents, and contaminated materials in designated biohazard bags and autoclave them before final disposal.
• Liquid Waste: Treat liquid waste, including culture media and wash solutions, with disinfectants before disposal.

Accident and Spill Response

• Spill Mercury Containment Kits: Have a spill kit on hand with absorbent materials, disinfectants, and personal protective equipment to manage accidental spills.
• Incident Reporting: Immediately report and document any spills, exposures, or accidents to the laboratory supervisor or biosafety officer.

Training and Competency

• Staff Training: Ensure all personnel are trained in biosafety protocols, bacterial handling, and emergency procedures.
• Ongoing Competency: Regularly update training and review safety procedures to maintain high standards of biosafety.

Documentation and Record Keeping

• Logbooks: Keep detailed records of all bacterial transformation activities, including the organisms used, procedures followed, and waste disposal methods.
• Biosafety Audits: Every laboratory where we made our work, conducts regular audits of laboratory practices and biosafety protocols to ensure compliance with established guidelines.

Regulations on Mercury Waste Management

Based on current regulations, there are no specific rules regarding mercury disposal. Therefore, it is governed by existing general regulations. Since 1995, mercury has been classified as a hazardous substance (DS 24176), and its management is subject to the Regulation for Activities with Hazardous Substances. Additionally, in DS 24782 Title VI, “On Hazardous Substances,” in Annex I lists mercury as part of the list of toxic compounds. Additionally, Article 54 of this regulation states that for mining activities, a form must be completed and submitted to the Ministry of the Environment for an Environmental License. Similarly, it indicates that hazardous substances must be treated in systems that eliminate, neutralize, or reduce their hazardous nature before final disposal.

In summary, the regulation does not address scenarios beyond mining activities, so the project developed by the iGEM Bolivia team adheres to the current regulations in the country.

List of Hazardous Substances in Mining Activities

49-sodium hydroxide	T,C
50-metallic magnesium	T
51-Mercury and its compounds	T
52-Methyl Isobutyl Ccarbinol	T
53-sodium nitrate	T
54-lead nitrate	T
55-Oxygen	E

Although Bolivian regulations on mercury spills may not be clear, we have implemented various containment measures based on laboratory biosafety protocols (Artisanal Gold Council, 2022). We briefly mentioned the mercury spill kit earlier, but we will now explain in more detail how this protocol was developed.

Handling Mercury Spills in the Laboratory

Handling spills in the laboratory requires training in basic procedures to follow in case such an incident occurs. For the iGEM Bolivia team, all members were trained through the “Biosafety Workshop.”

In the event of a mercury spill, the laboratory is equipped with all the necessary materials for spill management. Some of the available materials include:

Procedure:

1. Ventilate: Open windows and doors, and turn off the air conditioning.
2. Evacuate: Remove all personnel from the area where the experiments are being conducted.
3. Remove Metal Accessories: If wearing metal accessories, remove them as mercury reacts with metals.
4. Wear Protective Gear: Put on a mask, eye protection, and gloves.
5. Apply Sulfur: Use powdered sulfur to convert mercury into mercury sulfide.
6. Treat Broken Material: Ensure that any broken glass or debris also has sulfur applied to its surface.
7. Collect Glass: Use strips of cardboard to collect the broken glass and place absorbent paper towels over it.
8. Dispose of Absorbent Paper: Place the absorbent paper towels into a ziplock bag.
9. Ventilation: Keep the area ventilated for approximately 2 hours.
10. Dispose of PPE: Dispose of personal protective equipment (PPE) and mercury waste in a black trash bag.

To ensure that we meet all biosafety guidelines in our objective 3, once the prototype is ready to be used and contains the bacteria inside, the following waste disposal protocol will be followed.

Waste and Disinfection Protocol for the Mercury Biosensor Device

1. Preparation and Initial Handling

• Device Use: Before using the portable device for mercury concentration detection in waters, ensure that it is in optimal condition and that the transformed bacteria are properly contained in the covered bucket.
• Personal Protection: Throughout the handling process, wear personal protective equipment (PPE), It's especially important the use of gloves for the handling of the biosensor.

2. Disinfection and Waste Disposal Protocol
Completion of Use

• After completing the mercury detection, remove the device from the work area and carry out cleaning and disinfection in a designated biological waste handling area.

Handling the Bucket

• Disassembly: Remove the covered bucket from the device.

Once the container holding our genetically modified bacteria (GMOs) has been removed from the device, it will be placed in a specialized transport vessel. This vessel will then be sent to a nearby health station, where the following disinfection and disposal protocol will be carried out to ensure safe handling and environmental protection.

Within the health center, the following step will be carried out:

1. Disinfection and Waste Disposal Protocol for the Mercury Biosensor Device

Preparation and Initial Handling

• Device Usage: Before using the portable device for detecting mercury concentrations in water, ensure it is in optimal condition and that the transformed bacteria encapsulated in alginate beads are properly contained in the covered bucket.
• Personal Protection: Throughout the handling process, wear personal protective equipment (PPE). It is especially important to use gloves when handling the biosensor.

2. Disinfection and Waste Disposal Protocol

Completion of Use

• After completing the mercury detection, remove the device from the work area and perform cleaning and disinfection in a designated area for biological waste handling.

Handling the Bucket

• Disassembly: Remove the covered bucket from the device.
• Transport and Handling: Once the bucket containing the genetically modified organisms (GMOs) encapsulated in alginate is removed, place it in a specialized transport container. This container will be sent to a nearby health station, where the following disinfection and disposal protocol will be carried out to ensure safe handling and environmental protection.

Bacterial Inactivation

• Sterilization of the Bucket: Once the bucket arrives at the health center, it will be autoclaved at a temperature of 121°C for approximately 15 to 20 minutes for inactivation. After this time, the contents will be removed and the bacterial elimination process will be carried out, followed by the disinfection of the bucket.

Bacterial Disposal

Extraction:

• Preparation for Extraction: Before extracting the bacteria, set up a sterile work area with all necessary tools and materials. Ensure all personnel involved are using full PPE, including gloves, lab coats, and face shields, to minimize exposure risk.
• Sterile Tools: Use sterile, single-use tools such as pipettes, tweezers, or spatulas to extract the bacteria from the device. If reusable tools are used, ensure they are sterilized immediately before use. Work in a biosafety cabinet if available to further reduce contamination risk.
• Extraction Process: Carefully extract the bacteria from the device, minimizing the time they are exposed to the open air. Place the extracted bacteria directly into a designated biological waste container. If using pipettes, avoid creating aerosols by extracting and dispensing the bacterial culture slowly.

Bacteria Treatment:

• Containment: Immediately after extraction, seal the biological waste container to prevent accidental release of bacteria. The container should be clearly labeled with biohazard symbols and content details.
• Final Sealing: Once the bacteria have been inactivated and contained, securely seal the waste container and prepare it for disposal according to local regulations for biological waste. Ensure all steps are documented and that the container is handled only by authorized personnel.

Bucket Disinfection

Disinfectant Application

• Preparation: Before applying the disinfectant, ensure the area where disinfection will take place is well-ventilated and that all personnel are wearing appropriate PPE, including gloves, masks, and protective glasses.
• Disinfectant Selection: Use a disinfectant known to be effective against bacteria and mercury, such as a 10% sodium hypochlorite solution. Ensure the solution is freshly prepared or within its effective use period, as the potency of some disinfectants can decrease over time.
• Application Process: Carefully open the bucket and remove the encapsulated bacteria in alginate beads, discarding them in a biological waste container for future inactivation. Generously apply the disinfectant to all internal surfaces of the bucket, including the sides, bottom, and lid. Use a spray bottle or a clean cloth soaked in the solution to ensure even coverage. Pay special attention to seams, corners, and any cracks where contaminants might be trapped.
• Contact Time: Allow the disinfectant to act on the surfaces for the time specified by the manufacturer, usually 10 to 15 minutes. This is critical to ensure the disinfectant has enough time to kill all bacteria and neutralize any mercury residue. Do not rush this step, as insufficient contact time can lead to incomplete disinfection.

Rinsing

• Preparation for Rinsing: After the disinfectant has acted, prepare an adequate amount of sterile water for rinsing. Ensure the water is at room temperature to avoid thermal shock to the bucket material, which could compromise its integrity.
• Rinsing Process: Pour or spray sterile water into the bucket, ensuring all disinfectant residues are completely removed. Rotate and tilt the bucket to ensure all surfaces are rinsed, including hard-to-reach areas. Repeat the rinsing process as necessary to ensure no disinfectant residue remains.
• Drying: Place the bucket in a clean, contamination-free area, ideally in a well-ventilated space. Allow the bucket to air dry completely. To speed up drying, you may use a clean, lint-free cloth to gently dry the surfaces, but ensure the cloth does not introduce new contaminants. Verify that the bucket is completely dry before proceeding to the next step or storage.

Waste Disposal

Biological Waste

Waste Containers

• Segregation: All contaminated materials, such as bacterial waste, used gloves, absorbent paper, and disposable tools, should be placed in a designated biological waste container. This container should be clearly labeled with the biohazard symbol and content details to avoid confusion or improper handling.
• Handling Precautions: Handle the waste container with care, avoiding rough handling that could lead to spills or leaks. Ensure the container is not overfilled, as this could compromise the seal and increase exposure risk.

Device Disinfection

Device Cleaning:

• Preparation for Cleaning: Ensure the device is turned off, disconnected from any power source, and cooled if it was in use. Use appropriate PPE, including gloves and protective clothing, to avoid exposure to contaminants.
• Disinfection Procedure: Use an appropriate disinfectant (such as isopropyl alcohol solution) to clean the external surfaces of the device. It is important to apply the disinfectant first to a cloth and then clean the device with the damp cloth; do not apply disinfectant directly. For cleaning the device’s screen and buttons, use isopropyl alcohol in the same manner, applying it to a cloth and then to the screen (since some disinfectants can cause conductivity and short-circuit screens and buttons, which are the only electronic parts exposed to the user). Pay special attention to frequently touched areas or those that may have been exposed to contaminants during bucket handling.
• Multiple Applications: For maximum effectiveness, consider applying the disinfectant in multiple passes, allowing the surface to dry between applications. This helps ensure that any residual contaminants are effectively neutralized.

Inspection:

• Visual Check: After cleaning, visually inspect the device for signs of residue, moisture, or damage. Use a magnifying glass or bright light if necessary to check for cleanliness in small or intricate parts.
• Functional Check: If applicable, perform a basic functionality check to ensure the device operates correctly after cleaning. This might include checking buttons, screens, or connections to ensure no moisture or disinfectant remains in sensitive areas.
• Final Drying: Allow the device to air dry completely before storing or using it. Ensure no moisture remains, especially in areas where it could cause damage or affect device performance.

By implementing these measures, our protocol helps to protect the environment and ensures that the biosensor is used safely and responsibly.

References