Reliance on single-use plastics continues to harm the environment, as toxic chemicals from these materials leach into waterways, threatening plants, animals, and ecosystems. To help address this issue, I worked to develop a biodegradable plastic alternative capable of reducing environmental damage while maintaining similar performance properties. My objective was to construct and compare several plasmids to determine which design would be most effective for producing biodegradable plastics. Using Benchling, I selected compatible components from the Yarrowia lipolytica Golden Gate Toolkit to combine with my PHB genes. I chose a diverse set of genetic parts to give each plasmid the greatest chance of success. I referenced the publication “A Modular Golden Gate Toolkit for Yarrowia lipolytica Synthetic Biology” to guide my selections, although I found some of its information to be inconsistent. Once the plasmid designs were finalized, I assembled them using the Golden Gate assembly process, a method that allows multiple DNA fragments to be precisely joined in a single reaction. Each plasmid incorporated inserts from three genes, PhaA, PhaB, and PhaC, which encode enzymes essential for synthesizing polyhydroxybutyrate (PHB). PHB is a biodegradable polymer that can serve as a sustainable replacement for conventional plastics. In Benchling, I analyzed the gene sequences and designed forward and reverse primers for each insert. I then amplified the genes through PCR and confirmed successful amplification using gel electrophoresis. The construction process was complex and required multiple iterations before achieving consistent results. Once the plasmids were successfully assembled, I transformed them into competent cells and tested the resulting colonies for viability. I performed PCR on the colonies and confirmed that the DNA matched the expected lengths predicted in Benchling. With a verified plasmid, my next step will be to transform it into yeast and evaluate its ability to produce PHB, marking a promising advancement toward developing environmentally friendly alternatives to traditional plastics.
