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Safety

Safety in Lab

Since safety is an important aspect of the iGEM competition, we took extra caution to ensure that our experimental and device design had the least possible chance of posing a risk to the environment and general health and safety. The steps that we took are highlighted below.

Gearing up for Wetlab

Before we began any wetlab work for our project we all had to Environmental, Health and Safety (EHS) trainings which included:

COVID-19 Safety Training
EHS Laboratory Training Chemical and Biological
EHS Laboratory Training Standard Chemical
EHS Laser Safety Training for students who were to use the lasers.

We were also given an additional safety training which doubled as an orientation into wetlab where our laboratory supervisor, Dr. Alexis Stein, reviewed general lab rules including dress codes and waste disposal, sterile techniques, and general biology techniques. We were also given an overview of the procedure for reporting incidents in lab and what incidents need to be reported.

In addition, the team Laboratory & Safety Manager developed safety protocols for our lab space to include COVID protocols such as social distancing and mask wearing as well as general laboratory protocols including adequate labeling, dress codes, food policy and use of personal protective equipment (PPE) which we all agreed to follow.

Our next steps were to develop protocols for our experiments. We had to ensure that our experiments were safe to conduct and danger to the experimenter and environment was minimized. Once protocols were drafted, they were reviewed by the lab supervisor and edited where necessary. We were then trained and guided through the initial experiments to ensure that we were all comfortable with the procedures and the equipment.

Our general lab safety protocols were as follows:

Clean bench and work area before and after use
Thoroughly wash hands before and after experiments
Use the appropriate gloves, eye wear and lab coats for every experiment
Only wear long pants, closed toed shoes, solid T-shirts and tied hair to lab
No eating or drinking in the lab
Place waste in their appropriate disposal containers
Label all tubes appropriately with a descriptive label, date and initials

Our general experimental plan and safety protocols were also sent to our Environmental, Health and Safety Office for review to ensure that we were operating in compliance with the university and state safety regulations.

Experimental Safety

Some experiments that we designed had the potential of becoming unsafe, but since they were an essential part of our project, we had to figure out how to work around that. To do so we followed the ‘Hierarchy of Controls’ (Figure 1).

Figure 1: Hierarchy of Controls (The National Institute for Occupational Safety and Health)¹

A few of the measures taken are discussed below:

Chemically producing graphene oxide from graphite
The process of chemically making graphene oxide from graphite involves some caustic and toxic chemicals and generates a great amount of heat that may cause the reaction to become explosive. This involved highly concentrated sulfuric acid and nitric acid, and strong oxidizing agents, potassium permanganate and hydrogen peroxide. We employed engineering controls to reduce the risk of harm to our experimenter by conducting the experiment in a chemical fume hood with a transparent barrier. This experiment was also conducted in an ice bath to regulate the temperature and reagents were added slowly in small amounts to avoid explosions (Figure 2a). We also employed the use of PPE where our experimenter wore a flame resistant lab coat, acid/ base resistant gloves and splash-proof goggles (Figure 2b). Only glass material was used during this experiment including glass pipette tips to avoid dissolution and chemical spills.

Figure 2: a. Chemically producing graphene oxide from graphite in ice bath and chemical fume hood. b. Experimenter in flame resistant lab coat, splash-proof eyewear and acid/base resistant gloves.

Later in the project we opted to eliminate this process and its potential hazards by obtaining manufactured graphene oxide - Graphene Oxide Water Dispersion (Graphenea).
Chemically reducing graphene oxide
The common procedure for chemically reducing graphene oxide involves hydrazine.² Hydrazine is a very toxic substance which we did not want to work with. So we employed the substitution step in the Hierarchy of Controls (Figure 1) and found a safer alternative that would allow us to adequately reduce the graphene oxide, ascorbic acid.³
Waste disposal
Since we are working with bacteria although it is nonpathogenic, we did not want it to be released into the environment. We followed our waste disposal protocol where bacteria waste was collected in its own waste container and disposed of by the laboratory supervisor. Other liquid waste, including graphene oxide and reduced graphene oxide were collected in their own separate containers for appropriate disposal by the laboratory supervisor . We employed this method to be extra cautious since we do not know the implications of excess graphene oxide and reduced graphene oxide in waterways and the general environment. Experiments within our project involved the use of ethidium bromide and SYBR® Safe. Since ethidium bromide is carcinogenic and SYBR® Safe is a mild skin irritant that can be absorbed through skin, we used the appropriate PPE when handling these substances. Measures for handling included the use of latex gloves, avoiding contact with skin and eyes and ensuring that pipette tips and agarose gels containing these substances were collected in labeled containers for appropriate disposal by the laboratory supervisor.

Project Safety

We wanted to ensure that our project was in compliance with our department safety rules as well as iGEM safety rules. To ensure this, we did the following:

Chassis organism: in choosing our chassis organism, Shewanella oneidensis MR-1, and the other organism used throughout this project, Top 10 Escherichia coli, we ensured that they were Biosafety Level 1 (BSL-1) where both of these strains are non-pathogenic posing no known threat to human, plant or animal life.
Parts: we tried to choose parts from organisms that were BSL-1 and did not contribute to pathogenicty. All but one of our parts met this criteria where our outer membrane porin (oprF) was obtained from Pseudomonas aeruginosa which is a BSL-2 organism but since this part does not confer pathogenicity, we deemed it safe for use in our project.
Vectors: we selected a plasmid that was compatible with our chassis organism while ensuring that they did not confer any pathogenicty to our organism. We also ensured that our vector used a common antibiotic resistance gene. We therefore chose to use the vector pcD8 with kanamycin resistance (Keitz Lab, University of Texas Austin).⁴
Bacteria removal: to ensure that our final device did not contain any live bacteria that may interact with the environment, we developed a plan for washing out the S. oneidensis MR-1 from the microbial produced reduced graphene oxide. This washing process involved four sequential centrifugation washes with deionized water followed by overnight freezing at -80 C. We plated our washed reduced graphene oxide on TSB-agar plates and did not observe any S. oneidensis MR-1 growth. This indicated that our washing process was sufficient for getting rid of S. oneidensis MR-1 while maintaining the integrity of our reduced graphene oxide.

Hardware Safety

To manufacture our biosensing device, we used microfluidics, signal amplification, circuit design, and incorporating exchangeable electrodes.

For the circuit design, we used: breadboards, cables for breadboard, resistors, OpAmps, cables, alligator clips, oscillators, batteries, Arduino microcontrollers, electrodes.
For microfluidics, we used: 3D printer, device material (PDMS), sweat impermeable layer (glass).
For sleeve design, we used silicone and elastic/silicon/cotton strap.

The plan for each experiment was planned in the subteam (date, time of experiment, steps, safety measures). Circuit design involved working with Op Amps, resistors and Arduino microcontrollers. For Arduino, we used very low voltage (3V or 5V), which did not pose any danger to the members. We got trained on using an Arduino kit and learned how to connect all parts so that current is flowing in the right direction. Additionally, to introduce more electrical safety, we had each specific circuit plan checked over by our hardware teaching assistant. We also sketched circuits on Fritzing before we started building it.

Excessive input current over long periods of time or even short periods of time, if the current is high enough, can damage the op amp. To avoid this, we will create our protocols to use very low current (in mA).

PDMS was not hazardous and did not present any danger for members. It was stored in well-ventilated space and gloves were used when conducting experiments with this material.

Hierarchy of controls https://www.cdc.gov/niosh/topics/hierarchy/default.html (accessed Oct 19, 2021).
Marcano, D. C.; Kosynkin, D. V.; Berlin, J. M.; Sinitskii, A.; Sun, Z.; Slesarev, A.; Alemany, L. B.; Lu, W.; Tour, J. M. Improved Synthesis of Graphene Oxide. ACS Nano 2010, 4, 4806–4814.
Zhang, J.; Yang, H.; Shen, G.; Cheng, P.; Zhang, J.; Guo, S. Reduction of Graphene Oxide Vial-Ascorbic Acid. Chem. Commun. 2010, 46, 1112–1114.
Dundas, C. M.; Walker, D. J. F.; Keitz, B. K. Tuning Extracellular Electron Transfer by Shewanella Oneidensis Using Transcriptional Logic Gates. ACS Synthetic Biology 2020, 9, 2301–2315.

Team:Rochester/Safety