1. Did the team make a contribution to biosafety and/or biosecurity?

1. The wetlab team used malachite green as part of our PAPS kit and we made sure to handle and dispose of it properly. 

• Malachite green is a dye that can damage DNA. It is a known carcinogen and needs to be handled with care, and must be disposed of accordingly. 
• We followed proper procedure when using malachite green and after use, we collected all waste for proper disposal of the toxic substance. 

2. Our project used E. Coli in our project, so it was crucial that we maintained a thoroughly clean environment and avoided all avenues of possible contamination. 

• When growing our cultures we used antibiotics to stop any other micro bacteria from growing and to ensure we only grew the E. Coli we needed. 
• After all harvesting was done for cultures, all materials that came in contact with cultures were thrown away and disposed of as bio-hazard waste. This includes any solution that had resuspended pellets.  
• Additionally, after all lab work, all workspaces were wiped down thoroughly with ethanol. 

3. When running an SDS- PAGE Gel, fixing the gel is a crucial part of ensuring the samples remain in the gel during staining. While regularly used fixing solutions are harmful, we found a fixing solution that uses simple components that can be disposed of down the sink. We coined it the “Green” Fixing Solution due to its environmentally friendliness. 

• The “Green” fixing solution was a mix of 1 M Citric Acid and 5% Acetic Acid. 
• Acetic acid is a highly concentrated version of vinegar, and citric acid is normally found in sour candies. The mix of these two solutions allows for a safe and easily disposable fixing solution. 

4. When working in the lab, all wetlab members were trained on how to properly work in the lab. They were also taught the basic best practices and machine use throughout the lab.

• Anytime lab work needed to be done, there was at least one advisor/lab-trained mentor working alongside students in the lab to maintain lab safety and correct usage of lab equipment. 

2. Is their contribution well-characterized and/or well-validated?

1. The usage and disposal of malachite green is key to the lab workers' health. By using safe protocols, safety measures, and correct disposal, we limit the effect Malachite Green can have on the Team and the environment. This also teaches the wetlab team how to handle toxic materials which will provide a valuable learning opportunity as well as teach us good practice in a lab setting. 
2. By controlling the spread of E. Coli and limiting the opportunity for contamination, we maintain a clean and safe lab environment where everyone can conduct experiments in a controlled manner. BOSLab is a community lab and through the safe culturing measures we are able to keep it clean for everyone who uses the lab. 
3. Using the “Green” fixing solution allows us to follow the same protocol and still get good results without any of the harsh chemicals found in a normal fixing solution. As a community lab, we are always searching for cheaper and safer options for protocols and by finding this we can save a great deal of money on materials and disposal. 

3. Did the team build upon existing knowledge, understanding, tools or approaches? 

We were able to build upon our previous knowledge and approaches by utilizing the lab training we were taught and by modifying protocols to fit our needs and resources.

1. Modified protocols:
        - SDS Page Gel Fixing Solution
        - Using HEPES for PAPS Kit instead of Phosphatase Buffer
2. Machine usage in the lab:
        - Plate reader (620 nm) for PAPS Assay
        - PCR Machine → gPCR and Incubation
        - SDS Gel Runner
        - Electroporation Shocker

4. In addition to broader safety work, has the team managed risks from their project appropriately? 

Repetitive

5. Has the team addressed the use of synthetic biology in the real world?

The problem with current porcine-derived heparin is that it is unfractionated, which oftentimes increases the strength of the heparin product. That being said, unfractionated heparin (UFH) is very non-uniform and therefore unpredictable, as it causes an allergic reaction called heparin-induced thrombocytopenia (HIT). Fractionated heparin, more commonly called low molecular weight heparin (LMWH) is far more predictable. So, we decided to attempt to biosynthesize heparin in E. coli. We would then purify the heparin. By purifying, we would remove any and all contaminants and by synthetically producing the heparin, reducing the probability of contaminants because there are no live animals involved.  By using E. Coli, we know all possible contaminants in our sample and we can target them more accurately in our purification, making it better than the current porcine-derived heparin. Moreover, E. coli-produced heparin is more uniform than LMWH, so the risk of HIT is significantly lowered.