Team:Lambert GA/Safety
SAFETY
SAFETY
LABORATORY
TEAM SAFETY
- Wash in, wash out.
- 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 hair.
- Disinfect all surfaces with 70% ethanol before working.
- Disinfect all disposable tips, glassware, 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, 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 disposal of lab materials.
PROJECT SAFETY
Overview
AgroSENSE aims to make hydroponics, a sustainable form of agriculture, more accessible to small, urban communities by providing cost-effective and precise methods of sensing nutrient concentrations and environmental conditions. Utilizing biosensors, we aim to detect levels of specific nutrients (such as phosphate and nitrate) and the presence of harmful pathogens for plants.
The biosensors are designed to express different levels of GFP in response to nutrient volume presented in the aquaponics system. Additionally, the plant pathogen toehold riboregulators are designed to express GFP in the presence of the pathogen (trigger).
Specifically, we are continuing to characterize part BBa_K2447000 (phosphate sensor utilizing the native E. coli Pho Regulon) and part BBa_K1682018 (nitrate/nitrite sensor utilizing the traditional Nar Operon proteins). In addition, we are developing new toehold switches with their corresponding triggers to detect the presence of Phytophthora and Fusarium pathogens.
Wetlab
Nonpathogenic E. coli Chassis
We will use BL21, DH5-alpha, and 10-beta E. coli cells for transformations and cloning purposes only. BL21, DH5-alpha, and 10-beta E. coli are nonpathogenic and were developed for laboratory cloning use. The potential health and environmental hazards associated with BL21, DH5-alpha, and 10-beta E. coli are extremely limited and can be handled in Biosafety Level 1 laboratories.
Plans for Public Distribution of Biosensor Cells
As mentioned in our wet-lab pages, we are planning to implement AgroSENSE in the public by lyophilizing (i.e. freeze-drying) and creating cell-free extracts of our biosensor cells. As a proof-of-concept, we are looking into lyophilization of the phosphate and plant pathogen biosensor cells and distribution while accompanied with appropriate measures to dispose of the cells (e.g. UV light). For the nitrate sensor, the team is developing cell-free extracts in which public release poses significantly fewer biosafety concerns/issues.
Discarding Cells in the Field
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 [1].
Sonication Safety
In the preparation of cell-free lysates, sonication is used to lyse the cell membranes. Sonicators produce high frequency sound that can cause hearing damage [5], so earphone-type sound mufflers are worn during sonication to protect hearing.
Disposal of Sharps
During the dialysis step of cell-free lysate preparation, an 18-gauge needle is used to inject the lysate into the dialysis membrane. To safely dispose of the needle, it is sealed in a puncture resistant container before being disposed in a sharps container.
Hardware/Software
3D Printer
The frugal plate reader used to collect OD600 and fluorescence data from our nutrient biosensor cells is made using a 3D printer. The main safety hazard with our 3D printer is the temperature fluctuations. The nozzle heats up to 220 degrees Celsius. In addition, leaving the device unattended can be dangerous due to the 3D printer’s high temperatures that could cause a potential fire. However, our printer is advanced and possesses mechanisms to prevent this from happening, so the chance of this occurring is extremely rare, but if it occurs, it can cause serious damage and end up burning parts of the printer. The printer uses software called Crash Detection and Heat Overload to monitor the overall safety. A PI will continuously monitor the printer to prevent any accidents.
Electronic Devices
For this project, a significant amount of electronics work, and we have taken all of the proper safety precautions. Before any work with Arduino, Raspberry Pi, or related sensors and power supplies, team members ensure that they ground themselves with a large metal object. During any electronics work, team members use safety glasses. If working with motors or when soldering, team members wear electrically and thermally insulated gloves and work in a separate part of the room to reduce any risk of injury to other team members. Special precautions are taken when using any power supplies and AC adapters, including inspecting power supplies for damage before use, ensuring that proper voltages and currents are provided, and all liquids, food items, or other conductive materials are kept away.
Low-Pressure Systems and Vacuum Pumps
LyphoX, our frugal lyophilizer used to freeze-dry E. coli biosensors and cell-free lysates, requires the use of a 4 CFM (Cubic Feet per Minute) pump with a maximum pressure of 5 Pascals to maintain a low pressure for sublimation. The greatest concern when working with low-pressure systems is the implosion of containers which can splatter chemicals, shatter glass, and cause severe damage to surrounding areas. These hazards, however, can be minimized with appropriate personal protective equipment including chemical goggles, safety glasses, face shields, and explosion shields to protect against projectile matter. Special precautions should be taken to prevent water, solvents, or corrosive gases from entering the vacuum pump. The pumps are maintained with clean pump oil and will be replaced in the event of external damage. A PI will continuously monitor the lyophilizer to prevent any accidents.
Human Practices
Participants in all surveys provided signed consent for the release of their responses from themselves or a legal guardian. Participants in all events hosted by Lambert iGEM provided consent for photo and video release.
REGULATIONS
Lambert iGEM emphasized biosafety throughout the development of our proposed implementation. After designing our nutrient and plant pathogen biosensors, we addressed potential biosafety hazards by developing cell-free systems, thus eliminating the risk of releasing bacteria to the public. Additionally, in order to transport our biosensor cells and lysates to the public in a freeze-dried, or a completely deactivated state, we developed our frugal lyophilizer, LyphoX. We collaborated with stakeholders in academia, industry, and government to determine the optimal disposal methods and materials, including but not limited to UV lights, ethanol, and bleach. We aimed to explore how the different aspects of our biosensor implementation tie into governmental policies.
Lambert iGEM met with various members of the Georgia Department of Agriculture to understand the practicality of our biosensors from the government’s perspective. In an attempt to determine if there were any legislative barriers hindering us from distributing our biosensors commercially, we reached out to the Commissioner of the Georgia Department of Agriculture, Gary Black. A discussion with Commissioner Black led us to discover a gap in legislation regarding bioengineered products: regulatory parameters for genetically modified E. coli did not exist on the state-level. To gather more information, he directed us to his Food Safety and Laboratory Directors, Natalie Adan and John Shugart, respectively. Ms. Adan echoed her claims, stating that in the event of an E. coli outbreak, there are limited standardized protocols to eliminate the bacteria, leaving it up to the farmers to choose a method they deem best fit for their farm. As a team that works closely with E. coli, we understand the potential hazard bioengineered products pose to both farmers and consumers if handled incorrectly. Although our biosensors are nonpathogenic and present in chambers separated from actual produce, they pose a risk if mishandled. As bioengineered products are emerging within industry, we believe it is crucial to proactively address potential concerns instead of waiting for outbreaks to happen. This prompted us to work with a legal advisement team to develop legislation regarding the distribution and regulation of non-pathogenic recombinant biosensors into the agricultural market.
To commence this initiative, we contacted Ms. Ashley Haltom, the Vice President of Government Affairs at the Georgia BioEd Institute, to create a timeline and a list of action items for our legislation. She advised us to spend time researching the different opinions farmers had about the implementation of biosensors in their farms, including potential economic losses that may arise with legislative barriers. She also informed us about the timeline for the legislative process and encouraged us to reach out to our district’s representative, Chairman Todd Jones, a member of the Georgia House of Representatives, to present our proposal.
In our first meeting with the representative, we introduced our project and idea for biosafety legislation. We discussed the extensive timeline for developing, revising, introducing, discussing, and passing legislation. Upon consideration of our goals and timeline, Chairman Jones suggested that we shift our focus from drafting legislation to a regulation. He stated that the process of passing legislation is time-consuming, with the fastest creation time ranging from six months to two years. In addition, since there will likely be future developments in biosensor research, amendments to legislation would be required to adapt accordingly, and would be a similarly lengthy process. A pivot from legislation to regulation ensures that the governance of biosensors could be updated as often as necessary. Considering Mr. Jones' perspective, we returned to the Georgia BioEd Institute to restructure our plan.
Our next steps center around gathering feedback from the Atlanta Farmers Coalition, a networking group of over 30 farms from across the state, to determine potential concerns and factors to include in our regulation. We intend to use their responses to work with a legal advice team to draft a proposal of a regulation for the distribution of nonpathogenic recombinant biosensors to be introduced to the Georgia Department of Agriculture and in the Georgia House of Representatives.
LEAN6SIGMA
Background
During the fall semester of 2021, Lambert iGEM implemented Lean Six Sigma, an iterative organizational method that decreases turnaround time, delay time, and workplace hazards [2]. Which we adopted in an attempt to combat inefficiency in our laboratory to maximize our efficiency.
Methods
Lean Six Sigma is implemented in five distinct stages: define, measure, analyze, improve, and control (DMAIC). Our goal for this system was to organize lab equipment in a more intuitive manner to decrease time spent locating items as we are then able to optimize time and resources which would increase productivity around our project.
In our first iteration of the DMAIC process, the problem we were addressing was the lack of efficiency in our lab time that was limited due to members having to attend classes. To measure the extent of inefficiency in our lab, we spent time observing how much time team members spent locating reagents and equipment. After a close analysis, we found that due to the lack of organization, team members spent considerable amounts of time asking after the location of reagents and other needed items. To resolve this issue, we created a spreadsheet for lab storage (see Fig. 5). Each item was stored with the name of the item, the quantity, and shelf and row location. To organize lab equipment, we marked the designated lab equipment location with biosafety correlating color tape and used colored tapes in our pipette tip cabinet to differentiate between various tip sizes (see Fig. 1). In our shared refrigerator, we have a designated shelf and location (see Fig. 4) for each subcommittee as well as similarly labeled areas in our Laminar flow hood (see Fig. 3).
Figure 1. Pipette tip boxes were taped with designated colors according to tip sizes.
Figure 2. Heavy lab equipment marked with tape to show location permanence.
Figure 3. Left and right side of the laminar flow hood taped off to secure reagents and equipment in appropriate locations and maintain a clean environment.
Results:
Soon after adopting these corrections, there were immediate changes in our efficiency in our laboratory. For example, the taping in our Laminar flow hood, pipette tip cabinet, and around the laboratory resulted in an immediate decrease in time spent looking for reagents. Other modifications, such as our lab storage spreadsheet and refrigerator system took a more extended adjustment period to show results of effectiveness of adoption of this organization method. Once our laboratory implemented the protocol, we decreased preparation time as fewer members had to ask for the location of specific reagents and equipment.
Figure 4. Reagents were stored in alphabetical order.
Conclusion:
Lambert iGEM plans to continue implementing Lean Six Sigma in further iterations to increase productivity. By gradually adjusting lab protocols, we expect continual improvements in our lab.
Figure 5. Lambert iGEM used organized reagents and materials in our storage cabinets and recorded their quantities and locations.
REFERENCES
[1] Ballantyne, K. N., Salemi, R., Guarino, F., Pearson, J. R., Garlepp, D., Fowler, S., & van Oorschot, R. A. (2015). DNA contamination minimisation–finding an effective cleaning method. Australian Journal of Forensic Sciences, 47(4), 428-439.
[2] Inal, T. C., Goruroglu Ozturk, O., Kibar, F., Cetiner, S., Matyar, S., Daglioglu, G., & Yaman, A. (2018). Lean six sigma methodologies improve clinical laboratory efficiency and reduce turnaround times. Journal of Clinical Laboratory Analysis, 32(1), e22180.
[3] National Institute of Health. (2014). Biosafety and biosecurity in the United States. Federal Select Agent Program. Retrieved from https://www.nih.gov/sites/default/files/research-training/usg-safety-factsheet-2014.pdf
[4] Rochester Institute of Technology. (2019). 3-D Printer Safety. Retrieved from https://www.rit.edu/fa/grms/ehs/content/3-d-printer-safety.
[5] Sonicator Safety. UofR: EHS: Occupational Safety: Sonicator Safety. (n.d.). Retrieved October 22, 2021, from https://www.safety.rochester.edu/labsafety/sonicators.html.