430221 Protein Efflux Mapping in Single Living Cells with Synthetic Optical Nanosensors

Tuesday, November 10, 2015: 8:52 AM
Canyon C (Hilton Salt Lake City Center)
Markita Landry1, Hiroki Ando2, Allen Chen3, Vishal Kottadiel4, Timothy K. Lu5 and Michael S. Strano1, (1)Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, (2)Bioengineering, MIT, 02139, MA, (3)Bioengineering, MIT, Cambridge, (4)Rowland Institute, Harvard University, Cambridge, MA, (5)Massachusetts Institute of Technology, Cambridge, MA

Recent advances in metabolic engineering of Escherichia coli have provided an abundance of platforms for natural product biosynthesis and protein export [1]. However, real-time optical detection of E. coli metabolites remains a challenge. Previous work has shown great promise for the use of near-infrared fluorescent nanomaterials for optical detection of biomolecules [2, 3]. We present a platform for near-infrared optical detection of proteins exported from E. coli. By electrostatic conjugation of an aptamer-anchor polynucleotide sequence to a single-wall carbon nanotube, we are able to detect both RAP1 GTPase and HIV-1 integrase proteins, which each produce increases in near-infrared fluorescence of their respective aptamer-SWNT conjugates. Herein, we engineer the RAP1 gene, which is native to yeast, into E. coli. Subsequently, we monitor the productivity of RAP1 protein export by individual engineered E. coli cells over the course of 1 hour. We show that the process of induction, protein synthesis, and protein export is a highly stochastic process yielding variability in protein secretion on a single-cell level. We further show that our platform can be extended to the detection of protein from E. coli infected with T7 bacteriophages carrying the RAP1 gene. Our platform shows promise of extension for single-cell optical detection of a broad range of metabolic products with high spatial and temporal resolution.
  1. Keasling, J.D., Synthetic biology and the development of tools for metabolic engineering. Metabolic Engineering, 2012. 14(3): p. 189-195.
  2. Landry, M.P.*, Kruss, S.*, et al., Neurotransmitter Detection Using Corona Phase Molecular Recognition on Fluorescent Single-Walled Carbon Nanotube Sensors. Journal of the American Chemical Society, 2014. 136(2): p. 713-724.
  3. Landry, M.P.*, Zhang, J.Q.*, et al., Molecular recognition using corona phase complexes made of synthetic polymers adsorbed on carbon nanotubes. Nature Nanotechnology, 2013. 8(12): p. 959-968.

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