Assaying Interactions at the Ribosomal Tunnel in Vivo

In bacterial cells, ribosomes play a critical role in a myriad of cell functions including protein folding, protein transport across membranes, and stress responses. A current problem facing biochemists and chemical engineers alike is the ability to assess what other factors interact with ribosomes in vivo. Current methodologies employed rely heavily on purification techniques to isolate ribosomes along with their corresponding factors. A marriage of two techniques, Sec-M protein stalling paired with bimolecular fluorescence assay is used to create a stalled protein that would be capable of emitting photons of a certain wavelength when interacting with another tagged factor. Cells are analyzed via flow cytometry to confirm whether or not interaction is occurring at the protein tunnel.  In this work, positive and negative controls were created to collect baseline cytometric data to compare to tagged proteins thought to interact at the ribosomal tunnel.