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Construction of complex bacteriogenic protocells from living material assembly

Nature Protocols volume 20, pages 2586–2617 (2025)Cite this article

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Abstract

Protocell research offers diverse opportunities to understand cellular processes and the foundations of life and holds attractive potential applications across various fields. However, it is still a formidable task to construct a true-to-life synthetic cell with high organizational and functional complexity. Here we present a protocol for constructing bacteriogenic protocells by employing prokaryotes as on-site repositories of compositional, functional and structural building blocks to address this challenge. This approach is based on the capture and processing of two spatially segregated bacterial colonies within individual coacervate microdroplets to produce membrane-bounded, molecularly crowded, compositionally, structurally and functionally complex synthetic cells. The bacteriogenic protocells inherit sufficient biological components from their bacterial building units to exhibit highly integrated life-like properties, including biocatalysis, glycolysis and gene expression. The protocells can be endogenously remodeled to acquire diverse proto-organelles including a spatially partitioned nucleus-like DNA/histone-based condensate to store genetic material, membrane-bounded water vacuoles to adjust cellular osmotic pressure, a three-dimensional network of F-actin proto-cytoskeleton to support structural stability and proto-mitochondria to generate endogenous ATP as source of energy. The protocells ultimately develop a nonspherical morphology due to the continuous biogeneration of metabolic products by implanted living bacteria cells. This protocol provides a novel living material assembly strategy for the construction of functional protoliving microdevices and offers opportunities for potential applications in engineered synthetic biology and biomedicine. The protocol takes ~27 d to complete and requires expertise in microbiology, phase separation, biochemistry and molecular biology related techniques.

Key points

  • Membrane-bounded, molecularly crowded, compositionally, structurally and morphologically complex bacteriogenic protocells are constructed on the basis of the capture and on-site processing of spatially segregated bacterial colonies within individual coacervate microdroplets.

  • Bacteriogenic protocells are endogenously remodeled to acquire diverse proto-organelles and ultimately develop an amoeba-like nonspherical morphology.

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Fig. 1: Schematic view of the construction process of bacteriogenic protocells.
Fig. 2: Overview of the bacteriogenic protocell construction procedure (Steps 1–115).
Fig. 3: Overview of the bacteriogenic protocell construction procedure (Steps 116–144).
Fig. 4: Overview of the bacteriogenic protocell construction procedure (Steps 145–152).
Fig. 5: Construction of bacteriogenic protocells and evaluation of their cytomimetic functions.
Fig. 6: On-site augmentation and live cell energization of bacteriogenic protocells.

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Data availability

The main data discussed in this protocol are available in the supporting primary research paper17. All other data are available for research purposes from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank Y. Takebayashi and J. Spencer for help with bacterial cultures; H. Sun, C. Berger-Schaffitzel and E. Bragginton for help with gel electrophoresis and western blot analysis; A. Coutable and J. L. R. Anderson for providing the plasmid pEXP5-NT/deGFP; A. Leard from the Wolfson Bioimaging Facility for help with confocal imaging; and K. Heesom from the Proteomics Facility for proteomics analysis. Special thanks to Wolfson Bioimaging Facility for providing the microscopes. C.X. was funded by China 2024 Excellent Young Scientists Fund Program (Overseas) (20241B4435) and Shanghai Pujiang Program (23PJ1406000). N.M. and S.M. were funded by the ERC Advanced Grant Scheme (EC-2016-674 ADG 740235).

Author information

Authors and Affiliations

  1. School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai, P. R. China

    Can Xu, Mei Li & Stephen Mann

  2. Institute of Medical Robotics, Shanghai Jiao Tong University, Shanghai, P. R. China

    Can Xu

  3. Centre for Protolife Research and Centre for Organized Matter Chemistry, School of Chemistry, University of Bristol, Bristol, UK

    Mei Li & Stephen Mann

  4. Univ. Bordeaux, CNRS, Centre de Recherche Paul Pascal, Pessac, France

    Nicolas Martin

  5. Max Planck-Bristol Centre for Minimal Biology, School of Chemistry, University of Bristol, Bristol, UK

    Stephen Mann

  6. Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai, P. R. China

    Stephen Mann

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  1. Can Xu
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  2. Mei Li
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Contributions

C.X., M.L. and S.M. conceived the protocol design. C.X. and N.M. performed the experimental procedures. C.X. and M.L. prepared the figures. C.X. and S.M. wrote the manuscript. M.L. and N.M. performed the revision. S.M. supervised the work.

Corresponding authors

Correspondence to Can Xu or Stephen Mann.

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Nature Protocols thanks Yan Qiao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key reference

Xu, C. et al. Nature 609, 1029–1037 (2022): https://doi.org/10.1038/s41586-022-05223-w

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Statistical Source Data.

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Xu, C., Li, M., Martin, N. et al. Construction of complex bacteriogenic protocells from living material assembly. Nat Protoc 20, 2586–2617 (2025). https://doi.org/10.1038/s41596-025-01148-6

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