Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Poster Abstracts

26-POS Board 13 Tracking Orthogonal Fluorescent Ribosomes inside Living Cells Arvid H. Gynnå , Ivan L. Volkov, Johan Elf.Magnus Johansson. Uppsala University, Uppsala, Sweden.

The kinetic parameters of protein synthesis have been successfully studied by in vitro methods for decades. There are however many differences between the cellular environment and reconstituted systems that might affect these parameters. Among those are molecular crowding, confinement, uncertain intracellular ionic strength and pH, and possibly even the presence of yet unknown factors. Current methods for in vivo investigation of protein synthesis are limited. They are normally indirect, and/or give average numbers for several different molecular species. A method to directly measure the translation rate of individual ribosomes performing well-defined functions inside living cells is hence needed. To define the function of the ribosomes under observation inside a cell, a subset of the ribosomes should preferentially be orthogonal so they will only translate the mRNA of interest. In an organism with several rRNA loci, this can be achieved by modifying the anti-Shine-Dalgarno sequence of one of them and expressing a correspondingly orthogonal mRNA. For use in single-particle tracking experiments, the orthogonal ribosome subset must also be fluorescently labeled. We have developed such a system for E. coli , where the orthogonal small ribosomal subunits are labeled with an organic dye and thus can be monitored in singulo while operating inside the cell interacting with all natural components. This opens up the possibility to measure in vivo translation rates on defined mRNAs, as well as mapping the physical locations of the synthesis of different proteins.

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