Safety

Both marine and freshwater environments face enormous challenges--but these challenges have not been extensively addressed with synthetic biology approaches. Synthetic biology offers potential to address global problems associated with aquatic environments. Yet, few have been implemented in real-world aquatic environments. This is largely due to the significant gap in foundational knowledge of how engineered constructs function in real-world environments, which leads to insufficient safety understanding, which in turn results in a lack of effective policy. Our project addresses this knowledge gap to help promote the design of safe, effective synthetic circuits for eventual deployment in real world aquatic environments. As such, safety is always an overarching concern and goal of our project.

First, we emphasize that we are not deploying any organisms into the environment. We only use laboratory and simulated real-world conditions. To ensure that synthetic biology can apply its promise and potential to the real-world environments in which they will eventually be deployed, our project focuses on developing strategies to make engineered circuits and chassis to perform effectively in aquatic environments. Toward that end, we are employing a four-pronged approach to optimize the deployment of aquatic SynBio in diverse environments: 1. Software development for effective, safe chassis selection for specific environments; 2. Mathematical modeling to test the feasibility (with implications for safety) of synthetic biology approaches; 3. Meta-analysis of existing RNA-Seq databases to determine differences in gene expression between laboratory conditions and environmental conditions; 4. "Wet lab" case studies to perform controlled experiments to identify differences between laboratory and simulated real-world conditions. We will use the following as "case studies."

Oceans facing persistent issues with metal corrosion have become a critical global concern, leading to infrastructure failures and significant economic loss. We propose promoting the formation of thicker and denser biofilms by overexpressing three different regulatory pathways in Bacillus subtilis, comparing their effect on biofilm growth and persistence, and testing their ability to resist corrosion on metal.
In freshwater systems, harmful algal blooms (HABs) caused by cyanobacteria threaten aquatic biodiversity and release toxins hazardous to humans. We aim to use Microcystis aeruginosa to screen for novel cyanophages that could be engineered to treat HABs after formation. In addition, we will test Acinetobacter baylyi as a potential algicide.
At the household level, water distribution introduces new challenges—namely, biofilm formation in plumbing systems. To mitigate this, we propose engineering bacteriophages to deliver a gene encoding a functional cellulase into M. smegmatis after the formation of a biofilm, which will degrade the cellulose-rich extracellular matrix

We employ the following strains: Escherichia coli 10-beta; Bacillus subtilis 168 (ATCC 23857) Bacillus subtilis NCIB 3610 (ATCC 6051); Phage CrimD; Acinetobacter bayli. The cyanobacteria strain Microcystis aeruginosa strain 3037 (UTEX 3037, BSL1) will be used to screen for novel cyanophage from the environment. We plan on extracting DNA from Mycobacterium smegmatis 155 mc2 (ATCC 700084, BSL1) to extract the MSMEG_6752 gene for engineering into the phage for delivery, as well as using that same strain for phage infection and biofilm formation.

All organisms used are at the BSL 1 level, but we conduct all experiments in the entire lab at a BSL2 level to ensure optimal safety. Although the Microcystis aeruginosa (UTEX 3037, BSL1) we are working with are BSL1, we will be treating them as if they are BSL2 due to their expression of toxins. Lab members always wear gloves when conducting any wet lab work. Handwashing before departing the lab is always enforced. Lab coats are never shared. To prevent environmental hazards, such as environmental contamination from the lab, we will bleach and autoclave all BSL1 level waste. Although we do not have any known BSL2, this waste is collected and disposed of by our Environmental Health and Safety Office -- as are all toxic chemicals. At this point, we do not plan on using any BSL2 bacteria and plan to stay on the BSL1 level. BSL1 bacteria cannot cause disease in healthy humans but may be a danger for immunocompromised individuals. The bacteria could also cause a microbiome imbalance if released into the environment. Therefore, we will be taking strict precautions to prevent outside exposure. We do not plan on using our engineered constructs in the real world, but if the components of this project would be developed to the extent necessary, each component could be executed in different aquatic environments.

We do not plan on deploying our constructs outside of controlled lab environments due to legislative regulations as well as safety hazards, and it is outside the scope of this project. The B. subtilis engineered for corrosion prevention and the Nostoc engineered for erosion prevention would not be employed in the real world without proper genome integration of the pathway we are testing, since the plasmid-based approach includes antibiotic resistance, which could be transferred via horizontal gene transfer to other species of bacteria and would lack proper selection on a large scale. The engineered cyanophage would be employed to treat cyanobacterial algal blooms in freshwater, so the treatment would require full release. This would require extensive legislative approval and we do not plan on exploring this. The biosensor for predicting the onset of a harmful algal bloom would be used by collecting small samples from the environment and tested on a TX-TL solution. The phage treatment of biofilms in household water systems would need to be proved by the federal legislature and tested to prevent safety hazards to humans, so it is not plausible within our project.

We work closely with the William & Mary Environmental Health and Safety Office and William & Mary Institutional Biosafety Committee to ensure compliance with all federal, state, and university regulations regarding known hazards and potentially hazardous materials including recombinant DNA, infectious agents, toxic or otherwise dangerous reagents, and more. We will be consulting continually with W&M EH&S and W&M IBC. We also plan to consult with the Virginia Institute of Marine Science (VIMS) as part of our IHP to consult with experts on proper risk management. Our research complies with the William & Mary Chemical Hygiene Plan. Our team completed mandatory training with lessons with biosafety training and general radiation safety training based on our lab’s Institutional Biosafety Committee (IBC) protocol. We also completed mandatory readings of the Centers for Disease Control and Prevention (CDC) Manual and the National Institutes of Health (NIH) Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules.

Project-specific safety and security training helps to limit the risk of equipment damage and injuries while conducting this research. Additionally, this training lowers the risk of environmental damage as each team member is trained on how to properly dispose of BSL 1 trash and BSL 2 waste, as well as how to properly handle and mark hazardous material for safe disposal, which is based on our IBC protocol and the guidelines set in place by the William & Mary Environmental Health and Safety office. In addition to our personal safety, as we conduct research we discuss the potential impacts of our project (should it be further developed) to the environment and people. We plan to consult with experts and stakeholders about biocontamination as we conduct our IHP to ensure that we are taking the proper precautions, such as the Virginia Institute of Marine Science (VIMS). We are using exclusively BSL 1 level bacteria to ensure that there are minimized risks in our experimental design.

In terms of general laboratory safety, our entire team underwent extensive departmental safety training to minimize the risk of physical harm in the laboratory to the team and others. As per the guidelines of our University, all researchers must undergo this training annually. Materials that were covered include mechanical, chemical, radiation, and biosafety, as well as basic safety information such as attire, organization, hazard symbols, and prevention/handling of laboratory incidents. In addition, new lab members received ‘onboarding’ lab-specific training from our Principal Investigator, and acquired additional training from WM iGEM alum when learning new protocols. Our experimental and procedural protocols are all checked by our Principal Investigator before use, who is a member of WM IBC and is the safety coordinator for our lab (William and Mary’s Bioengineering Lab) and the third floor of the Integrated Science Center (the building in which our lab is located). We also confirm our methods of trash and waste disposal with our co-Principal Investigator, who is on the Emergency Management Committee. Our protocols all contain detailed instructions and alert members to possible safety risks (for example, lab members are instructed to use the fume hood when creating agarose gels with EtBr). Additionally, members must attend specific training sessions with our Molecular Core Facility Manager (Vincent Roggero) when using certain equipment, such as autoclaves and the ultracentrifuge. These various forms of training will help minimize risks associated with laboratory work and equipment. Finally, our team members receive ongoing safety training for every protocol we employ.