Abstract
Single-molecule force spectroscopy enables mechanical testing of individual proteins, but low experimental throughput limits the ability to screen constructs in parallel. We describe a microfluidic platform for on-chip expression, covalent surface attachment and measurement of single-molecule protein mechanical properties. A dockerin tag on each protein molecule allowed us to perform thousands of pulling cycles using a single cohesin-modified cantilever. The ability to synthesize and mechanically probe protein libraries enables high-throughput mechanical phenotyping.
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Change history
05 November 2014
In the version of this article initially published, the grant "European Research Council Grant Cellufuel (Advanced Grant 294438)" was mistakenly left out of the Acknowledgements. The error has been corrected in the HTML and PDF versions of the article.
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Acknowledgements
M.O. is grateful to the Elite Network of Bavaria (IDK-NBT) for a doctoral fellowship. M.A.N. acknowledges support from Society in Science—The Branco Weiss Fellowship administered by the ETH Zürich. The authors acknowledge support from the DFG Sonderforschungsbereich 1032 and the European Research Council Grant Cellufuel (Advanced Grant 294438). The authors thank E. Bayer (Weizmann Institute) for starting genetic materials used for Doc and Coh modules.
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M.O., M.A.N. and H.E.G. designed the research; M.O., W.O., M.A.J. and T.V. performed experiments; D.A.P. helped with immobilization strategies; M.O., W.O., M.A.J., L.F.M. and M.A.N. performed data analysis; M.O., W.O., M.A.J., M.A.N. and H.E.G. cowrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Microfluidic chip overview.
(a) Photograph of a microfluidic chip bonded to a glass slide with a US dime for scale. Control channels are filled with food dye for better visualization. (b) Pattern of a typical DNA array, consisting of repeats of rows with four different genes and one row with nothing spotted as negative control. (c) Photograph of a bonded PDMS chip onto the glass slide with DNA spots in the back chamber. The orange highlighted frame shows a zoom in of the bottom left corner. (d) Typical fluorescence collage assembled from 640 single fluorescence micrographs of each protein chamber on one single chip shows pattern of expressed protein (assembly not to scale). Fluorescence signal of TagRFP reveals expression levels and Dockerin specificity. Here, low passivation of the protein chamber facilitates visualization. (e) Three of 640 adjacent dumbbell-shaped chambers, one with sfGFP DNA spotted (left), one with Xylanase DNA (center) and one negative control without DNA (right). Control channels are visualized with food dye: neck valve (green), sandwich valve (red), and button valve (blue). (f) Fluorescence images showing GFP signal (top) from expressed and immobilized ybbR-sfGFP-Dockerin (left), ybbR-Xylanase-Dockerin (center) with negative control lacking the spotted DNA (right). The bottom row shows the signal from the TagRFP detection construct, which specifically bound to the Dockerin tag via the Cohesin domain.
Supplementary Figure 7 Exemplary force traces
Example curves showing (a) uninterpretable interaction, (b) non-specific interaction of cantilever with surface, (c) no interaction, and (d) a specific Xylanase-Dockerin unfolding and unbinding trace. Curves similar to those shown in a-c were excluded from the analysis.
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Otten, M., Ott, W., Jobst, M. et al. From genes to protein mechanics on a chip. Nat Methods 11, 1127–1130 (2014). https://doi.org/10.1038/nmeth.3099
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DOI: https://doi.org/10.1038/nmeth.3099
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