Following iGEM's Rules and Policies

We devised our project with the iGEM safety rules as an underlying principle and conducted our wet lab while duly adhering to all safety procedures delineated by the competition:

1. “Release Beyond Containment” Policy: Our team did not release, utilize, or deploy any sources or products of genetically modified organisms (e.g., Synechocystis sp. ATCC27184) outside the lab.
2. “White List” Policy: Our project did not involve using any part of organisms from Risk Group 3 or 4, nor any other animals, parts, or materials outside the limits of the Whitelist designated by iGEM. Our project does not involve using the SARS-CoV-2 virus in any part of the procedure.
3. “No Human Experimentation” Policy: Our project does not involve testing on humans, including our team members. No part of our procedure involved the use of humans or human samples, including bodily specimens or psychological outcomes in any manner.
4. “Animal Use” Policy: Our project does not involve the use of animals or animal samples, including vertebrates and higher-order invertebrates, in any part of our experiment.
5. “Human Subjects Research” Policy: Our team did not conduct any surveys, interviews, or other human subjects research within the context of wet lab experiments.
6. “Antimicrobial Resistance” Policy: Our project does not involve creating novel antimicrobial resistance factors or increasing antimicrobial resistance in any organism. All procedures of our wet lab were carried out under the regulation of the iGEM policy.
7. “Gene Drives” Policy: Our wet lab did not involve gene drives in any part of the procedure.
8. “Coronavirus” Policy: As aforementioned, our wet lab does not involve the SARS-CoV-2 virus in any form in all procedure parts.
9. According to iGEM's risk group classification, our workspace's biosafety level is Level 2 (moderate containment). As such, it consists of a cell culture room and laboratory lecture space. It also contains an exposed bench and biosafety cabinet.

About Our Lab

Our wet lab was conducted over multiple months to ensure the accuracy and precision of our results. The wet lab can be distinctly divided into three general processes: creating the culture media for our bacteria to capture and convert CO₂ to produce PHB directly, staining Synechocystis cells with Nile Red for visualization of PHB underneath fluorescent microscopes, and next-generation sequencing of Synechocystis cells. Throughout our lab work, safety requirements were carefully considered and met in a multitude of aspects:

• Wearing closed-toe shoes, gloves, and lab coats
• Tying long hair back to reduce chemical exposure
• Discarding contaminated tubes and pipette tips immediately
• Informing ourselves of emergency protocols
• Disinfecting and sterilizing our lab environment
• Not consuming foods or beverages in the lab

About Our Project

Project Goal

Our project aims to engineer Synechocystis sp. ATCC27184 (a cyanobacterium) to absorb atmospheric CO₂ and enhance the production of polyhydroxybutyrate (PHB), a bioplastic material. We utilized adaptive evolution techniques to select strains that thrive in high pH culture conditions. We also utilized Next-Generation Sequencing (NGS) methods to analyze DNA and gene expression levels to better understand how the selected bacteria increased PHB production. This would help address two major global issues simultaneously. By improving CO₂ absorption, our engineered cyanobacteria can help reduce greenhouse gas levels, slowing global warming. Additionally, producing PHB offers a sustainable alternative to petroleum-derived plastics.

Engineering and Experimental Approach

• Genetic Modifications: Our project aims to genetically modify the Synechocystis sp. ATCC27184 to enhance its PHB production capabilities. This involves restricting the population to bacteria capable of effectively converting CO₂ to PHB and increasing its population.
• DNA/RNA Usage: We did not utilize or synthesize DNA or RNA in our experiments. Instead, our project focused on in silico experiments to identify candidate gene alterations for improving the efficiency of CO₂ to PHB bioconversion. Rather than conducting direct experiments, we employed directed evolution techniques like NGS methods to analyze genetic modifications and determine their effectiveness.
• Safety & Resource Overview: Our project’s genetic and chemical components are non-hazardous. We ensured that each aspect of our experiments followed the established safety guidelines and did not use any AI tools in its development.

Identifying Project Risks

The hazard potentially occurs because we are going to use the following chemical substances for the bacteria culture media: sodium nitrate (NaNO₃), potassium phosphate (K₂HPO₄), magnesium sulfate (MgSO₄), calcium chloride (CaCl₂), citric acid, ferric ammonium citrate, ethylenediaminetetraacetic acid (EDTA), sodium carbonate (Na₂CO₃), trace metals mix (e.g., zinc sulfate, copper sulfate, cobalt nitrate, sodium molybdate). The components listed above are generally not classified as hazardous in the concentrations typically used in the BG11 medium. However, specific components, like EDTA and some trace metals, in higher concentrations could pose hazards when exposed to the skin or eyes:

Anticipating Future Risks

Although our project is foundational and we do not have a specific real-world application, it could be applied in a factory or other industrial manufacturing context.

Possible Future Uses

Suppose our project on the evolutionary engineering of Synechocystis sp. for high-efficiency CO₂ to PHB bioconversion is successful. In that case, it will open up several transformative opportunities across various industries and environmental sectors:

• 1) Sustainable Bioplastics Production:
  • Developing Synechocystis strains with enhanced CO₂ to PHB conversion efficiency will provide a sustainable and eco-friendly bioplastic production method. PHB (polyhydroxybutyrate) is a biodegradable polymer that can replace conventional plastics, reducing plastic pollution and reliance on fossil fuels.
• Carbon Sequestration:
  • High-efficiency CO₂ conversion by engineered Synechocystis can be used as a biological tool for carbon sequestration. This can help mitigate the effects of climate change by capturing and converting atmospheric CO₂ into valuable bioproducts, thus contributing to global efforts to reduce greenhouse gas concentrations.

Future Risks

My project’s future development would not require release beyond the containment: Accidental exposure to a hazardous organism or chemical might harm human health and safety. We would use biocontainment strategies to prevent the spread.

• Physical containment, which involves using physical barriers to prevent the spread of the organism, will be used. For example, engineered Synechocystis can be kept in sealed photobioreactors, which prevent their release into the environment.

Managing Risks

The laboratory manager and safety team will support our project in managing risks and hazards. We will strictly follow the standard NIH safety rules to store our Synechocystis bacteria safely in an appropriately registered space. Although all of our team members have learned about safety precautions before working in the lab, we will continue to emphasize the importance of following safety rules. We have learned safety rules regarding lab access, biosafety level differences, biosafety equipment, proper microbial technique, sterilization of workspace, emergency procedures, chemical/fire safety, physical biosecurity, and personnel biosecurity.

Our team has implemented project-specific safety training to manage any risks that may come our way. Overall, our expert support, strict rule enforcement, training, and well-established procedures will aid in managing the risks in our project by guaranteeing that all members have sufficient comprehension of safety protocols. Implementing these measures will significantly reduce the potential for accidents, contamination, and biohazards.