Biophysical Society Thematic Meeting - June 28-July 1, 2015

New Biological Frontiers Illuminated by Molecular Sensors and Actuators

Monday Speaker Abstracts

Optomechanical Actuators for Controlling Mechanotransduction in Living Cells Khalid Salaita 1,2 . 1 Emory University, Atlanta, USA, 2 Georgia Institute of Technology & Emory University, Atlanta, GA, USA. See abstract: Pos-37 Board 37 Synthetically Rerouting Phagocytosis by Rapidly Turning Inert Cells into “Eat You” Mode Toru Komatsu 1,4 , Hiroki Onuma 1 , Tetsuo Nagano 2 , Yasuteru Urano 1,3 .Takanari Inoue 5 . 1 The University of Tokyo, Tokyo, Japan, 3 The University of Tokyo, Tokyo, Japan, 4 JST PRESTO, Tokyo, Japan, 5 Johns Hopkins University, Baltimore, MD, USA. 2 The University of Tokyo, Tokyo, Japan, See Abstract: Pos-19 Board 19 Prokaryotes lack membranous organelles to compartmentalize biochemical reactions. To optimize the efficiency of engineered metabolic pathways in E.coli, artificial organelles based on porous protein shells and synthetic RNA/protein scaffold have been proposed to bring metabolic enzymes to close proximity and thus speed up reactions. In this study, we aim to develop, in E.coli, a unique protein scaffold to spatially assemble pathways of interest, based on heterologous expression of Caulobacter crescentus proteins, PopZ and SpmX. We have cloned and standardized both PopZ and SpmX genes from Caulobacter crescentus, and created fluorescent fusions to validate their localization and capability as molecular scaffold in E.coli. We scanned different expression levels of PopZ and established stable formation of PopZ complex in E.coli. We found that SpmX remained diffused throughout when it was singly expressed in E.coli. When SpmX was co-expressed with PopZ, it then co-localized with PopZ foci, suggesting that there is direct interaction between PopZ and SpmX. We then tested if SpmX can serve as the adaptor for PopZ to bring different proteins of interest to close proximity. As a proof of concept, we performed bimolecular fluorescence complementation. No fluorescence was observed when these two split fluorescent fusion proteins were co-expressed. Fluorescent foci was detected when PopZ is present, strongly suggesting that PopZ complex can serve as a molecular scaffold to spatially assemble pathways via SpmX∆C adaptors. With this scaffold device, we plan to assemble and optimize the efficiency of a foreign metabolic pathway in E.coli. We anticipate this system to be user-friendly in wide range of microbial metabolic engineering applications. Organization of Intracellular Reactions with Heterologous Protein Scaffold Hsiao-Chun Huang . National Taiwan University, Taipei, Taiwan.

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