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Programming multi-protein assembly by gene-brush patterns and two-dimensional compartment geometry

Nature Nanotechnology volume 15, pages 783–791 (2020)Cite this article

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Abstract

The assembly of protein machines in cells is precise, rapid, and coupled to protein synthesis with regulation in space and time. The assembly of natural and synthetic nanomachines could be similarly controlled by genetic programming outside the cell. Here, we present quasi-two-dimensional (2D) silicon compartments that enable programming of protein assembly lines by local synthesis from surface-immobilized DNA brushes. Using this platform, we studied the autonomous synthesis and assembly of a structural complex from a bacteriophage and a bacterial RNA-synthesizing machine. Local synthesis and surface capture of complexes provided high assembly yield and sensitive detection of spatially resolved assembly intermediates, with the 3D geometry of the compartment and the 2D pattern of brushes dictating the yield and mode of assembly steps. Localized synthesis of proteins in a single gene brush enhances their interactions, and displacement of their genes in separated brushes leads to step-by-step surface assembly. This methodology enables spatial regulation of protein synthesis, and deciphering, reconstruction and design of biological machine assembly lines.

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Fig. 1: Protein synthesis and assembly in 2D compartments.
Fig. 2: Solution assembly and surface scaffolding in 1D layouts.
Fig. 3: The effect of gene brush layout on wedge assembly.
Fig. 4: Deciphering assembly line order.
Fig. 5: Gene composition and compartment geometry impact E. coli RNAP assembly.
Fig. 6: Local regulation of gene expression by resource partitioning.

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Data availability

The data necessary to interpret, replicate and build upon this work appears in the article and its Supplementary information. Additional data can be made available from the corresponding authors upon reasonable request.

Code availability

The information needed for the computer simulation in Fig. 2 appears in the article and its Supplementary information.

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Acknowledgements

We acknowledge funding from the Israel Science Foundation (grant no. 1870/15), the United States–Israel Binational Science Foundation (grant no. 2014400), and the Minerva Foundation (grant no. 712274) for the work on the T4 wedges. We thank the Office of Naval Research (award no. N62909-18-1-2094) for funding the work on RNAP assembly. We thank M. Levy for discussions.

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Author notes

  1. These authors contributed equally: Ohad Vonshak, Yiftach Divon.

Authors and Affiliations

  1. Department of Chemical and Biological Physics, The Weizmann Institute of Science, Rehovot, Israel

    Ohad Vonshak, Yiftach Divon, Shirley S. Daube & Roy H. Bar-Ziv

  2. Max Planck Institute of Colloids and Interfaces, Potsdam, Germany

    Stefanie Förste, Reinhard Lipowsky & Sophia Rudorf

  3. School of Physics and Astronomy, University of Minnesota, Minneapolis, MN, USA

    David Garenne & Vincent Noireaux

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Contributions

O.V., S.S.D., R.H.B.-Z. and V.N. conceived the cell-free assembly of phages. O.V., Y.D., S.S.D. and R.H.B.-Z. designed the experiments and analysed the data. O.V. and Y.D. performed the experiments. D.G. and V.N. provided the cell-free E. coli extract. O.V., S.F., S.R. and R.L. did the modelling and computational work. O.V., Y.D., S.F., S.R., S.S.D. and R.H.B.-Z. wrote the manuscript. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Shirley S. Daube or Roy H. Bar-Ziv.

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Extended data

Extended Data Fig. 1 Competition between on-chip solution and scaffolded assembly.

a, Scheme, co-expression and interaction of gp10 and gp7 in solution prior to surface binding sequester the scaffolding of gp7 onto gp10 pre-bound to the surface. b, Dose response of gene-10 fraction in a mixed DNA brush with fixed amount of gene-7. Surface gp11 traps were pre-bound with gp10. Surface bound gp10-7 complexes revealed by post-staining with FL-gp8. Detailed gene composition of all experiments appears in Supplementary Table 3. Individual data points and mean values are shown, error bars represent ± s.d. The number of samples for each data point (b) is listed in Supplementary Table 4.

Supplementary information

Supplementary Information

Supplementary information on computational modelling, Supplementary Figs. 1–11 and Tables 1–4.

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Vonshak, O., Divon, Y., Förste, S. et al. Programming multi-protein assembly by gene-brush patterns and two-dimensional compartment geometry. Nat. Nanotechnol. 15, 783–791 (2020). https://doi.org/10.1038/s41565-020-0720-7

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