Tori Guarino
Tori Guarino headshot
Year: 2023
Faculty Advisor: Laura Gunn
Mentor: Zhen Guo Oh
Mentor: Bryce Askey

Optimizing an E. coli expression system for Penium margaritaceum Rubisco

In photosynthesis, the enzyme Rubisco catalyzes the fixation of carbon dioxide (CO2) into carbohydrates. To reduce Rubisco’s ability to react with oxygen, some photosynthetic organisms have evolved CO2-concentrating mechanisms (CCMs) which increase CO2 concentrations around Rubisco above normal atmospheric levels. Most green algae possess a pyrenoid-based CCM, consisting of a Rubisco-containing organelle in the chloroplast into which CO2 is pumped. Penium margaritaceum is an appealing algal species for pyrenoid study due to its unique evolutionary position as a sister species to land plants. Components identified from study of its pyrenoid should be more easily transferable to crop plants. In this study, to aid the engineering of a P. margaritaceum pyrenoid into a land plant, we develop and optimize an E. coli expression system for P. margaritaceum Rubisco. The triple plasmid system used within the E. coli expression has one plasmid for the large and small subunits that come together to form the Rubisco molecule, one for chaperonins that assist with folding of the large subunit, and one for chaperones that assist with further assembly of the large subunit. As for my experimentation, there have been large and small subunits produced, but the purified protein samples also contain large amounts of chaperonins, signaling that there is a component of the E. coli system that is not allowing the large subunit to be folded properly. More research is required in order to find the core set of chaperonins and chaperones needed for optimal expression of the P. margaritaceum Rubisco system.

During this Research Experience for Undergraduates at Boyce Thompson Institute, I heavily expanded upon my lab techniques, bioinformatics, and plant biology curiosity. In the Gunn lab, I had the opportunity to execute tasks that I have never had the opportunity to do, such as run protein and DNA gels, create PCR products, transform bacteria, create competent cells, and immerse myself within microbiology, just to name a few. These lab techniques further allowed me to gain confidence within the wet lab environment. The lab community was also a meaningful part of my experience. My mentor, PI, and other REU students created such a safe and inquisitive space to not only conduct research, but to grow as a person. I learned greatly about my potential as a researcher as well as became enlightened about future career paths, such as graduate school and beyond, all within the appreciation of flora.