Results

Results:

Initially, our project focused on utilizing the MEP (Methylerythritol Phosphate) metabolic pathway in E. coli because this pathway naturally produces geranyl pyrophosphate (GPP), a key universal precursor in the biosynthesis of all terpenes.

Our goal was to incorporate the limonene synthase gene (LimS-GPPS) into the E. coli system. If the engineered bacteria produced limonene, a cyclic terpene commonly used in fragrances and as a precursor in various chemical processes, the limonene would be secreted from the cells as a gas, making it relatively easy to collect and measure.

In addition to limonene, also we aimed to engineer the production of vanillin, a key compound used in flavoring and perfumery. Vanillin biosynthesis can be achieved in E. coli by introducing two genes: fcs (feruloyl-CoA synthetase) and ech (enoyl-CoA hydratase/aldolase). These genes enable the conversion of ferulic acid—a natural plant metabolite—into vanillin through a fermentation process. This pathway provided an efficient method to produce vanillin in a microbial host.

Initially, we believed that expressing both limonene and vanillin in E. coli would be relatively straightforward, so we began exploring additional modifications to further enhance our bacterial system. During our research, we discovered that terpene biosynthesis could be regulated by environmental factors, including light. This led us to consider light-regulated gene expression, which would allow us to control terpene production in response to external stimuli.

Through further investigation, we learned about a promising blue-light inducible gene regulation system called pDawn, developed by a professor specializing in optogenetics. The pDawn plasmid system enables precise control of gene expression in E. coli using blue light as a trigger. Excited by the possibility of integrating this cutting-edge technology into our project, we reached out to the Dr. Lou at the Shenzhen Institute for Synthetic Biology who connected us with Professor Jin. We successfully obtained samples of the pDawn plasmid to incorporate into our experimental design.

The process of obtaining the pDawn plasmid as well as the synthesis and delivery of the gene limonene and vanillin constructs required for terpene production took much longer than expected due to external delays. As a result, we were unable to begin the key phase of our wet lab experiments. Compounding this issue, our school was suddenly permanently closed at the end of July. The sudden uncertainty and stress that our team members were confronted with of having to find new schools to attend and the fact that when we found new schools for the new academic year that started in September, we were scattered into different schools made it almost impossible to continue with the project. Consequently, we were unable to make as much progress as we had initially hoped on the experimental aspects of the project, particularly in the wet lab.

Despite these setbacks, the foundational work we accomplished — gaining knowledge of synthetic biology, exploring light-regulated gene expression systems, and setting the stage for terpene biosynthesis in E. coli — provided us with valuable insights and prepared us for future experimentation and to take our knowledge to our new schools. Hopefully we can inspire our new schools to start IGEM programmes if they do not have any already.