Organisms
We are using Escherichia coli and Synechococcus elongatus, both of which are classified as BSL1 by the ATCC and have been approved for use in this project by IIT IBC, our institutional biosafety committee. These organisms are acting as our chasses to hold a modified gene that expresses PETase, an enzyme which dissolves PET plastic.
No significant risks are posed by these organisms. E. coli K12 strains are a non-pathogenic variant of normal intestinal flora in healthy individuals, and have been extensively used in laboratories throughout the world precisely due to their low risk level. Generally, cyanobacteria (blue-green algae) are free living, non-pathogenic photosynthetic organisms already found in most environments. S. elongatus is a common environmental cyanobacteria that is found in waterways. Indeed, the specific strain we are using was collected from UTEX Culture Collection of Algae, which is hosted in a creek near the University of Texas.
Experiments
To produce PET nanoparticles for our experiments, trifluoroacetic acid, or TFA, was used. TFA is a particularly strong acid, posing notable risks to any users. To mitigate these risks, the use of TFA in the laboratory required strict adherence to PPE requirements. This includes protective glasses, two layers of gloves, and full lab coat and splash apron.
Ethidium bromide, a known carcinogen, is used to make the gel through which DNA moves during electrophoresis. To confront these risks, it is required that everyone exposed to ethidium bromide in the lab be wearing safety goggles and gloves when gel is being synthesized.
Aside from this, none of our experiments pose any significant dangers to our team and do not involve hazardous chemicals or organisms. The core experiment involves modifying an existing polyethylene terephthalate hydrolase (PETase) enzyme, which degrades PET plastic. We will then clone it into a vector compatible with expression in our selected cyanobacteria, a pAM series plasmid. This vector carried both an E coli and a cyanobacterial origin of replication, and is thus functional in both organisms. This will then be evaluated in an assay where the degradation of plastic microparticles exposed to the experimental cyanobacteria will be measured by DLS, fluorescence, or other assay methods. We will synthesize PET nanoparticles form post consumer PET (soda bottles) to be as similar to environmental plastics as possible, except that we will be labeling them with fluorescein to aid in detection.
Environment
There would likely be some objection to the introduction of genetically modified organisms into the environment, as there is a heated debate about the safety and ethics of GMOs. Though GMOs have not shown significant safety risk in any studies, their use is still controversial. Thus, these concerns should be addressed regardless.
A similar discussion of potential risks is with regards to the introduction of non-native species into an ecosystem. This is a problem frequently faced as a side effect of human activities, and can devastate a biome by upsetting the natural balance of its native species. In our case, however, this is a somewhat more complex consideration. We specifically chose cyanobacteria for this purpose because it is already native to the ocean ecosystem. However, since the s. elongatus cyanobacteria will be genetically modified, its degradation of PET plastic could be a disruptive behavior. Though the presence of PET and other plastics in the ocean is already a disturbance to the natural ecosystem, altering the system once again to undo these changes is, in itself, another disruption, and may have unintended and unpredictable consequences.
We have no intent or plans to release such organisms to the wild, and all cultures with this plasmid in it will be sterilized by autoclaving before disposal. This scope of this project is simply a demonstration of feasibility, and we hope it prompts debate about how to deal with this important environmental problem.