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


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.

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.


  1. 1.

    Gu, H., Chao, J., Xiao, S.-J. & Seeman, N. C. A proximity-based programmable DNA nanoscale assembly line. Nature 465, 202–205 (2010).

    CAS  Google Scholar 

  2. 2.

    Douglas, S. M. et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414–418 (2009).

    CAS  Google Scholar 

  3. 3.

    Bath, J. & Turberfield, A. J. DNA nanomachines. Nat. Nanotechnol. 2, 275–284 (2007).

    CAS  Google Scholar 

  4. 4.

    Leung, K., Chakraborty, K., Saminathan, A. & Krishnan, Y. A DNA nanomachine chemically resolves lysosomes in live cells. Nat. Nanotechnol. 14, 176–183 (2019).

    CAS  Google Scholar 

  5. 5.

    Douglas, S. M., Bachelet, I. & Church, G. M. A logic-gated nanorobot for targeted transport of molecular payloads. Science 335, 831–834 (2012).

    CAS  Google Scholar 

  6. 6.

    Schwarz-Schilling, M. et al. Optimized assembly of a multifunctional RNA-protein nanostructure in a cell-free gene expression system. Nano Lett. 18, 2650–2657 (2018).

    CAS  Google Scholar 

  7. 7.

    Freeman, R. et al. Reversible self-assembly of superstructured networks. Science 362, 808–813 (2018).

    CAS  Google Scholar 

  8. 8.

    Strackharn, M., Pippig, D. A., Meyer, P., Stahl, S. W. & Gaub, H. E. Nanoscale arrangement of proteins by single-molecule cut-and-paste. J. Am. Chem. Soc. 134, 15193–15196 (2012).

    CAS  Google Scholar 

  9. 9.

    Daube, S. S. & Bar-Ziv, R. H. Protein nanomachines assembly modes: cell-free expression and biochip perspectives. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 5, 613–628 (2013).

    CAS  Google Scholar 

  10. 10.

    Shieh, Y.-W. et al. Operon structure and cotranslational subunit association direct protein assembly in bacteria. Science 350, 678–680 (2015).

    CAS  Google Scholar 

  11. 11.

    Holt, C. E., Martin, K. C. & Schuman, E. M. Local translation in neurons: visualization and function. Nat. Struct. Mol. Biol. 26, 557–566 (2019).

    CAS  Google Scholar 

  12. 12.

    Minton, A. P. Implications of macromolecular crowding for protein assembly. Curr. Opin. Struct. Biol. 10, 34–39 (2000).

    CAS  Google Scholar 

  13. 13.

    Daube, S. S., Arad, T. & Bar-Ziv, R. Cell-free co-synthesis of protein nanoassemblies: tubes, rings, and doughnuts. Nano Lett. 7, 638–641 (2007).

    CAS  Google Scholar 

  14. 14.

    Asahara, H. & Chong, S. In vitro genetic reconstruction of bacterial transcription initiation by coupled synthesis and detection of RNA polymerase holoenzyme. Nucleic Acids Res. 38, e141 (2010).

    Google Scholar 

  15. 15.

    Matthies, D. et al. Cell-free expression and assembly of ATP synthase. J. Mol. Biol. 413, 593–603 (2011).

    CAS  Google Scholar 

  16. 16.

    Rustad, M., Eastlund, A., Jardine, P. & Noireaux, V. Cell-free TXTL synthesis of infectious bacteriophage T4 in a single test tube reaction. Synth. Biol. 3, ysy002 (2018).

    CAS  Google Scholar 

  17. 17.

    Bracha, D., Karzbrun, E., Daube, S. S. & Bar-Ziv, R. H. Emergent properties of dense DNA phases toward artificial biosystems on a surface. Acc. Chem. Res. 47, 1912–1921 (2014).

    CAS  Google Scholar 

  18. 18.

    Daube, S., Bracha, D., Buxboim, A. & Bar-Ziv, R. H. Compartmentalization by directional gene expression. Proc. Natl Acad. Sci. USA 107, 2836–2841 (2010).

    CAS  Google Scholar 

  19. 19.

    Heyman, Y., Buxboim, A., Wolf, S. G., Daube, S. S. & Bar-Ziv, R. H. Cell-free protein synthesis and assembly on a biochip. Nat. Nanotechnol. 7, 374–378 (2012).

    CAS  Google Scholar 

  20. 20.

    Levy, M., Falkovich, R., Daube, S. S. & Bar-Ziv, R. H. Autonomous synthesis and assembly of a ribosomal subunit on a chip. Sci. Adv. 6, eaaz6020 (2020).

    Google Scholar 

  21. 21.

    Karzbrun, E., Tayar, A. M., Noireaux, V. & Bar-Ziv, R. H. Programmable on-chip DNA compartments as artificial cells. Science 345, 829–832 (2014).

    CAS  Google Scholar 

  22. 22.

    Tayar, A. M., Karzbrun, E., Noireaux, V. & Bar-Ziv, R. H. Synchrony and pattern formation of coupled genetic oscillators on a chip of artificial cells. Proc. Natl. Acad. Sci. USA 114, 11609–11614 (2017).

    CAS  Google Scholar 

  23. 23.

    Efrat, Y., Tayar, A. M., Daube, S. S., Levy, M. & Bar-Ziv, R. H. Electric-field manipulation of a compartmentalized cell-free gene expression reaction. ACS Synth. Biol. 7, 1829–1833 (2018).

    CAS  Google Scholar 

  24. 24.

    Shin, J., Jardine, P. & Noireaux, V. Genome replication, synthesis, and assembly of the bacteriophage T7 in a single cell-free reaction. ACS Synth. Biol. 1, 408–413 (2012).

    CAS  Google Scholar 

  25. 25.

    Garamella, J., Marshall, R., Rustad, M. & Noireaux, V. The all E. coli TX-TL toolbox 2.0: a platform for cell-free synthetic biology. ACS Synth. Biol. 5, 344–355 (2016).

    CAS  Google Scholar 

  26. 26.

    Yap, M. L. et al. Role of bacteriophage T4 baseplate in regulating assembly and infection. Proc. Natl Acad. Sci. USA 113, 2654–2659 (2016).

    CAS  Google Scholar 

  27. 27.

    Yap, M. L. et al. Sequential assembly of the wedge of the baseplate of phage T4 in the presence and absence of gp11 as monitored by analytical ultracentrifugation. Macromol. Biosci. 10, 808–813 (2010).

    CAS  Google Scholar 

  28. 28.

    Bray, D. & Lay, S. Computer-based analysis of the binding steps in protein complex formation. Proc. Natl Acad. Sci. USA 94, 13493–13498 (1997).

    CAS  Google Scholar 

  29. 29.

    Roy, R. D., Rosenmund, C. & Stefan, M. I. Cooperative binding mitigates the high-dose hook effect. BMC Syst. Biol. 11, 74 (2017).

    Google Scholar 

  30. 30.

    Murugan, A. et al. Undesired usage and the robust self-assembly of heterogeneous structures. Nat. Commun. 6, 6203 (2015).

    CAS  Google Scholar 

  31. 31.

    Luke, K. et al. Microarray analysis of gene expression during bacteriophage T4 infection. Virology 299, 182–191 (2002).

    CAS  Google Scholar 

  32. 32.

    Marshall, R. & Noireaux, V. Quantitative modeling of transcription and translation of an all-E. coli cell-free system. Sci. Rep. 9, 11980 (2019).

    Google Scholar 

  33. 33.

    Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755 (2001).

    CAS  Google Scholar 

  34. 34.

    Mathew, R. & Chatterji, D. The evolving story of the omega subunit of bacterial RNA polymerase. Trends Microbiol. 14, 450–455 (2006).

    CAS  Google Scholar 

  35. 35.

    Hillebrecht, J. R. & Chong, S. A comparative study of protein synthesis in in vitro systems: from the prokaryotic reconstituted to the eukaryotic extract-based. BMC Biotechnol. 8, 58 (2008).

    Google Scholar 

  36. 36.

    Erijman, A., Dantes, A., Bernheim, R., Shifman, J. M. & Peleg, Y. Transfer-PCR (TPCR): a highway for DNA cloning and protein engineering. J. Struct. Biol. 175, 171–177 (2011).

    CAS  Google Scholar 

  37. 37.

    Kincade, J. M. & deHaseth, P. L. Bacteriophage lambda promoters pL and pR sequence determinants of in vivo activity and of sensitivity to the DNA gyrase inhibitor, coumermycin. Gene 97, 7–12 (1991).

    CAS  Google Scholar 

  38. 38.

    Buxboim, A., Daube, S. S. & Bar-Ziv, R. Ultradense synthetic gene brushes on a chip. Nano Lett. 9, 909–913 (2009).

    CAS  Google Scholar 

  39. 39.

    Caschera, F. & Noireaux, V. Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription–translation system. Biochimie 99, 162–168 (2014).

    CAS  Google Scholar 

  40. 40.

    He, B. et al. Rapid mutagenesis and purification of phage RNA polymerases. Protein Express. Purif. 9, 142–151 (1997).

    CAS  Google Scholar 

  41. 41.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    CAS  Google Scholar 

  42. 42.

    Buxboim, A. et al. A single-step photolithographic interface for cell-free gene expression and active biochips. Small 3, 500–510 (2007).

    CAS  Google Scholar 

Download references


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.

Author information




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.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation


Quick links

Find nanotechnology articles, nanomaterial data and patents all in one place. Visit Nano by Nature Research