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Nat Chem Biol. 2018 Apr;14(4):352-360. doi: 10.1038/s41589-018-0004-9. Epub 2018 Feb 26.

A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters.

Author information

1
Media Lab, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA.
2
Howard Hughes Medical Institute, Department of Neurobiology, Harvard Medical School, Boston, MA, USA.
3
Department of Electrical Engineering and Computer Science, MIT, Cambridge, MA, USA.
4
Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, MA, USA.
5
Simons Center Data Analysis, Simons Foundation, New York, NY, USA.
6
Department of Mechanical Engineering, MIT, Cambridge, MA, USA.
7
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
8
Picower Institute for Learning & Memory and Department of Brain & Cognitive Sciences, MIT, Cambridge, MA, USA.
9
Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, Cambridge, MA, USA.
10
Department of Neurobiology, Faculty of Life Sciences, University of Vienna, Wien, Austria.
11
Department of Cell Biology and Neuroscience, Montana State University, Bozeman, Montana, USA.
12
Department of Biological Physics, Eotvos University, Budapest, Hungary.
13
Media Lab, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA. esb@media.mit.edu.
14
Department of Biological Engineering, MIT, Cambridge, MA, USA. esb@media.mit.edu.
15
MIT Center for Neurobiological Engineering, MIT, Cambridge, MA, USA. esb@media.mit.edu.
16
Department of Brain and Cognitive Sciences, MIT, Cambridge, MA, USA. esb@media.mit.edu.
17
MIT McGovern Institute for Brain Research, MIT, Cambridge, MA, USA. esb@media.mit.edu.

Abstract

We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.

PMID:
29483642
PMCID:
PMC5866759
DOI:
10.1038/s41589-018-0004-9
[Indexed for MEDLINE]
Free PMC Article

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