Transient expression of cyanobacterial b-carboxysome proteins in Nicotiana to study protein assembly

β-carboxysomes, a type of microcompartment present in most photosynthetic cyanobacteria, concentrates CO2 near Rubisco inside a protein shell to improve carbon fixation in photosynthesis.  Incorporation of β-carboxysomes into C3 crop plants could potentially improve their photosynthetic efficiency and increase their overall yield.  Approximately ten types of proteins are proposed to form the full carboxysome, but little is known about their individual structures and roles in carboxysome function.  This study focused on possible assemblies formed by various carboxysomal proteins when expressed within tobacco chloroplasts.  Carboxysomal shell proteins, CcmP, CcmO, and CcmK2, and interior protein CcmM35 were fluorescently tagged and attached to a chloroplast transit peptide using overlap extension PCR, and the gene inserts were ligated into vectors for E. coli transformation.  Agrobacteria were then transformed with the vector constructs, and the genes were transiently expressed in Nicotiana tabacum and N. benthamiana using agroinfiltration.  The leaf tissues were imaged using confocal microscopy to determine the proteins’ distribution within the chloroplast.  A diffuse fluorescent signal, spread over the entire chloroplast, gives evidence that a protein is not involved in the assembly of macromolecular complexes with other proteins.  Presence of punctate signals suggests that a transiently expressed protein possibly associates with other proteins to form a significant assembly. Further understanding of the assembly of carboxysomal proteins is helpful for eventually attaining full carboxysome construction within plant chloroplasts.

My Experience

This internship has benefited me in so many ways and has been great preparation for my future career.  I learned many new research techniques while working on my project and heard about many others through the weekly research talks given by BTI faculty.  Especially from these talks I have a much better idea of what is going on in the field and where I could possibly focus my work in the future.  As well as other professional skills applicable to any job, my scientific writing and presentation skills have grown, and I feel more confident about proposing and presenting a research project.  I have also learned a lot about graduate school from the faculty and students here.  I have had a fantastic summer meeting and working with great scientists and great people, and I feel confident and excited about going to graduate school for plant science in the future.