Analyzing and Inquiring
In the analyzing and inquiring phase of the design cycle we reviewed scientific literature in order to be able to move forward on our project of transforming PET plastic to ethanol. Among the pivotal scientific papers that guided our methodology were: “Characterization and engineering of a two-enzyme system for plastics depolymerization”, “Improving ethylene glycol utilization in Escherichia coli fermentation”, and “Metabolic engineering of Escherichia coli for production of mixed-acid fermentation end products”. These scientific papers collectively provided us with a foundation, guiding our decisions on enzyme systems to engineer our biological parts.
In addition, we also sought to understand the real-world impact of plastic pollution, specifically in our local community of Peru. Recognizing the environmental consequences, including land contamination, harm to wildlife, and damage to biodiversity, served as a compelling motivation for our project.
Developing ideas
At the beginning of the project, our main objective was to contribute to our local community with the biodegradation of plastic. During this crucial phase of the design cycle, we carefully evaluated several approaches, including the use of plastic-digesting fungi and the modification of Ideonella sakaiensis. In the end, after careful consideration, we chose to modify Escherichia coli (E. coli), mainly due to its well-established characteristics and suitability for genetic modification. However, much of the information and analysis carried out was useful for the future.
Additionally, our exploration revealed an innovative application for the plastic decomposition process: ethanol production. This biofuel could play a vital role in our community by serving as a valuable resource to fuel ovens in isolated communities, offering an alternative and sustainable energy source.
Creating the solution
The first step to creating the solution was the making of the biological constructs. In order to do this, we had already identified possible genes that we could use, bu we had to organize them in a pathway that would lead from PET to ethanol. In addition, we had to identify a concise selection of genes that would be suitable for their incorporation into plasmids due to the limitations of our high school laboratory environment. The genes we chose were, PETase, MHETase, aldA, fucO, and AdhE. The image of the pathway can be seen below.
We organized these genes into constructs, including essential components such as promoters, ribosome binding sites (RBS), genes facilitating protein expression, and a reporter gene. The latter would express chromoproteins in distinct colors, depending on the construct. The biological parts added to the registry were BBa_K4881027, BBa_K4881028, and BBa_K4881029.
Evaluating
A positive aspect of our evaluation is the fact that in our second transformation, we were able to obtain transformants of our constructs. We were also able to redo the transformation of our chloramphenicol backbone plasmid, O11 from the iGEM distribution kit (BBa_J04450), which would be the plasmid that will act as the chloramphenicol backbone for our third construct. since we were unable to obtain transformed cells in our first attempt to transform them.
After obtaining transformed cells, we attempted to perform DNA extraction and gel electrophoresis to evaluate the uptake of our plasmids by E. coli cells. However, we were not able to obtain the majority of lines that indicated that the DNA we were using was completely transformed in the cell. Therefore, due to time limitations, we decided to carry out a bioassay with the samples we had, demonstrating that cells with Construct 1 are capable of degrading plastic. One aspect to improve is the amount of liquid media, cells, and sample size in our bioassay so that the results can be more accurate and more clearly demonstrate the functionality of the plasmid.
Troubleshooting
