Non-Pathogenic Chassis

We consciously selected non-pathogenic strains of yeast (S. cerevisiae) and bacteria (Dh5α and BL21 E. coli) as our chassis organisms. By using microorganisms generally recognized as safe, we minimize the risk of accidental infections or harm to humans, animals, or plants.

Safe Genetic Parts

Our choice of genetic parts and components is based on their proven safety record. We have carefully screened and selected genetic elements that are known to be non-toxic and non-harmful, reducing the possibility of unintended negative consequences. The plasmids we are using contain safe insert protein-coding genes, all naturally occurring enzymes: HpaBC, D-LDH, TAL, 4CL, TyrB, and rosmarinic acid synthase (RAS).

The functions of these enzymes are as follows:

• HpaBC genes express a 4-hydroxyphenylacetate 3-monooxygenase reductase complex. This complex converts 4-hydroxyphenylpyruvate into 4-dihydroxyphenyllactate by adding a hydroxyl group to the benzene ring.
• D-LDH genes express lactate dehydrogenase which carries a reduction reaction, converting a carbon double-bonded oxygen to a carbon-bonded hydroxyl group to convert 4-dihydroxyphenyllactate into salvianic acid A.
• TyrB genes express tyrosine aminotransferase. This protein converts 4-hydroxyphenylpyruvate into L-tyrosine. The TAL genes express tyrosine ammonia lyase. This protein converts L-tyrosine into p-coumaric acid.
• 4CL genes express 4-coumaroyl CoA-ligase. This protein converts caffeic acid to caffeoyl CoA.
• RAS genes express rosmarinic acid synthase. This protein fuses caffeoyl CoA and salvianic acid A to produce rosmarinic acid.

Substituting with Safer Materials:

In our proof-of-concept experiments, we have taken steps to replace potentially dangerous materials with safer alternatives. For instance, we have opted for non-toxic hydrogel materials that are biocompatible, ensuring that any accidental contact or exposure poses minimal risk. Additionally, we have decided to synthesize rosmarinic acid as our proof of concept because this molecule does not pose a significant threat to humans and has very low toxicity. Our original idea was to synthesize a medical compound such as papaverine, but that could pose a threat since they are regulated molecules and should not be synthesized outside of authorized facilities that have better means of containment.

Personal Protective Equipment (PPE):

Team members are required to wear appropriate PPE, including lab coats and gloves, to minimize the risk of exposure to chemicals and biological materials. Additionally, team members are required to wear safety goggles when working on hardware and when they perform organic chemistry procedures such as preparing the HPLC samples and handling strong acids. Fume hoods were also used when handling strong acids, fumes, or boiling liquids.

Sterile Techniques:

We maintained strict sterile techniques to prevent contamination when handling microorganisms, including the modified yeast and bacteria used in our project. To discard liquid contaminated samples, they were mixed with bleach to a concentration of 10%, then diluted with water and poured in the sink. Solid contaminated waste was autoclaved before disposal.

Chemical Handling:

Chemicals are handled with care, following established protocols for storage, disposal, and dilution. We are mindful of the potential hazards of chemicals used in our experiments.

Waste Disposal:

The University of Rochester has a rigorous waste disposal system in place to ensure that all biohazardous and chemical waste is properly disposed of according to regulations.