Construction of complex bacteriogenic protocells from living material assembly

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. Access through your institution Buy or

subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through your institution Access Nature and 54 other Nature Portfolio journals Get

Nature+, our best-value online-access subscription $29.99 / 30 days cancel any time Learn more Subscribe to this journal Receive 12 print issues and online access $259.00 per year only

$21.58 per issue Learn more Buy this article * Purchase on SpringerLink * Instant access to full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout

ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS LIVING MATERIAL ASSEMBLY OF

BACTERIOGENIC PROTOCELLS Article 14 September 2022 PROGRAMMED SPATIAL ORGANIZATION OF BIOMACROMOLECULES INTO DISCRETE, COACERVATE-BASED PROTOCELLS Article Open access 08 December 2020 A DE

NOVO MATRIX FOR MACROSCOPIC LIVING MATERIALS FROM BACTERIA Article Open access 21 September 2022 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. REFERENCES *

Dzieciol, A. J. & Mann, S. Designs for life: protocell models in the laboratory. _Chem. Soc. Rev._ 41, 79–85 (2012). Article  CAS  PubMed  Google Scholar  * van Stevendaal, M. H. M. E.,

van Hest, J. C. M. & Mason, A. F. Functional interactions between bottom-up synthetic cells and living matter for biomedical applications. _ChemSystemsChem_ 3, e2100009 (2021). Article 

Google Scholar  * Jeong, S., Nguyen, H. T., Kim, C. H., Ly, M. N. & Shin, K. Toward artificial cells: novel advances in energy conversion and cellular motility. _Adv. Funct. Mater._ 30,

1907182 (2020). Article  CAS  Google Scholar  * Toparlak, O. D. & Mansy, S. S. Progress in synthesizing protocells. _Exp. Biol. Med._ 244, 304–313 (2018). Article  Google Scholar  *

Yewdall, N. A., Mason, A. F. & van Hest, J. C. M. The hallmarks of living systems: towards creating artificial cells. _Interface Focus_ 8, 20180023 (2018). Article  PubMed  PubMed

Central  Google Scholar  * Kurihara, K. et al. Self-reproduction of supramolecular giant vesicles combined with the amplification of encapsulated DNA. _Nat. Chem._ 3, 775–781 (2011). Article

  CAS  PubMed  Google Scholar  * Dora Tang, T. Y. et al. Fatty acid membrane assembly on coacervate microdroplets as a step towards a hybrid protocell model. _Nat. Chem._ 6, 527–533 (2014).

Article  PubMed  Google Scholar  * Li, M., Harbron, R. L., Weaver, J. V. M., Binks, B. P. & Mann, S. Electrostatically gated membrane permeability in inorganic protocells. _Nat. Chem._

5, 529–536 (2013). Article  PubMed  Google Scholar  * Rodríguez-Arco, L., Li, M. & Mann, S. Phagocytosis-inspired behaviour in synthetic protocell communities of compartmentalized

colloidal objects. _Nat. Mater._ 17, 857–863 (2017). Google Scholar  * Qiao, Y., Li, M., Booth, R. & Mann, S. Predatory behaviour in synthetic protocell communities. _Nat. Chem._ 9,

110–119 (2016). PubMed  Google Scholar  * Koga, S., Williams, D. S., Perriman, A. W. & Mann, S. Peptide–nucleotide microdroplets as a step towards a membrane-free protocell model. _Nat.

Chem._ 3, 720–724 (2011). Article  CAS  PubMed  Google Scholar  * Huang, X. et al. Interfacial assembly of protein–polymer nano-conjugates into stimulus-responsive biomimetic protocells.

_Nat. Commun._ 4, 2239 (2013). Article  PubMed  Google Scholar  * Dora Tang, T. Y., van Swaay, D., deMello, A., Ross Anderson, J. L. & Mann, S. In vitro gene expression within

membrane-free coacervate protocells. _Chem. Commun._ 51, 11429–11432 (2015). Article  CAS  Google Scholar  * Li, M., Green, D. C., Anderson, J. L. R., Binks, B. P. & Mann, S. In vitro

gene expression and enzyme catalysis in bio-inorganic protocells. _Chem. Sci._ 2, 1739–1745 (2011). Article  CAS  Google Scholar  * Walde, P. & Ichikawa, S. Enzymes inside lipid

vesicles: preparation, reactivity and applications. _Biomol. Eng._ 18, 143–177 (2001). Article  CAS  PubMed  Google Scholar  * Walde, P., Goto, A., Monnard, P.-A., Wessicken, M. & Luisi,

P. L. Oparin’s reactions revisited: Enzymic synthesis of Poly(adenylic acid) in micelles and self-reproducing vesicles. _J Am. Chem. Soc._ 116, 7541–7547 (1994). Article  CAS  Google

Scholar  * Xu, C., Martin, N., Li, M. & Mann, S. Living material assembly of bacteriogenic protocells. _Nature_ 609, 1029–1037 (2022). Article  CAS  PubMed  Google Scholar  * Mann, S.

Systems of creation: the emergence of life from nonliving matter. _Acc. Chem. Res._ 45, 2131–2141 (2012). Article  CAS  PubMed  Google Scholar  * Xu, C., Hu, S. & Chen, X. Artificial

cells: from basic science to applications. _Mater. Today_ 19, 516–532 (2016). Article  CAS  Google Scholar  * Martino, C. & deMello, A. J. Droplet-based microfluidics for artificial cell

generation: a brief review. _Interface Focus_ 6, 20160011 (2016). Article  PubMed  PubMed Central  Google Scholar  * Solé, R. V., Munteanu, A., Rodriguez-Caso, C. & Macía, J. Synthetic

protocell biology: from reproduction to computation. _Philos. Trans. R. Soc. Lond. B_ 362, 1727–1739 (2007). Article  Google Scholar  * Jaffe, J. D. et al. The complete genome and proteome

of _Mycoplasma_ mobile. _Genome Res._ 14, 1447–1461 (2004). Article  CAS  PubMed  PubMed Central  Google Scholar  * Thornburg, Z. R. et al. Fundamental behaviors emerge from simulations of a

living minimal cell. _Cell_ 185, 345–360.e328 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Williams, D. S. et al. Polymer/nucleotide droplets as bio-inspired functional

micro-compartments. _Soft Matter_ 8, 6004–6014 (2012). Article  CAS  Google Scholar  * Weibull, C. The isolation of protoplasts from _Bacillus megaterium_ by controlled treatment with

lysozyme. _J. Bacteriol._ 66, 688–695 (1953). Article  CAS  PubMed  PubMed Central  Google Scholar  * Birdsell, D. C. & Cota-Robles, E. H. Production and ultrastructure of lysozyme and

ethylenediaminetetraacetate-lysozyme spheroplasts of _Escherichia coli_. _J. Bacteriol._ 93, 427–437 (1967). Article  CAS  PubMed  PubMed Central  Google Scholar  * van den Bogaart, G.,

Guzman, J. V., Mika, J. T. & Poolman, B. On the mechanism of pore formation by melittin. _J. Biol. Chem._ 283, 33854–33857 (2008). Article  PubMed  PubMed Central  Google Scholar  *

Pandidan, S. & Mechler, A. Nano-viscosimetry analysis of the membrane disrupting action of the bee venom peptide melittin. _Sci. Rep._ 9, ARTN 10841 (2019). Article  Google Scholar  *

Oberholzer, T., Wick, R., Luisi, P. L. & Biebricher, C. K. Enzymatic RNA replication in self-reproducing vesicles—an approach to a minimal cell. _Biochem. Bioph. Res. Co._ 207, 250–257

(1995). Article  CAS  Google Scholar  * Oberholzer, T., Albrizio, M. & Luisi, P. L. Polymerase chain-reaction in liposomes. _Chem. Biol._ 2, 677–682 (1995). Article  CAS  PubMed  Google

Scholar  * Ishikawa, K., Sato, K., Shima, Y., Urabe, I. & Yomo, T. Expression of a cascading genetic network within liposomes. _Febs. Lett._ 576, 387–390 (2004). Article  CAS  PubMed 

Google Scholar  * Carlson, E. D., Gan, R., Hodgman, C. E. & Jewett, M. C. Cell-free protein synthesis: applications come of age. _Biotechnol. Adv._ 30, 1185–1194 (2012). Article  CAS 

PubMed  Google Scholar  * Khosla, C. & Keasling, J. D. Metabolic engineering for drug discovery and development. _Nat. Rev. Drug Discov._ 2, 1019–1025 (2003). Article  CAS  PubMed 

Google Scholar  * Skruzny, M. et al. Molecular basis for coupling the plasma membrane to the actin cytoskeleton during clathrin-mediated endocytosis. _Proc. Natl Acad. Sci. USA_ 109,

E2533–E2542 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Emelyanov, V. V. Mitochondrial connection to the origin of the eukaryotic cell. _Eur. J. Biochem._ 270, 1599–1618

(2003). Article  CAS  PubMed  Google Scholar  * Vellai, T. & Vida, G. The origin of eukaryotes: the difference between prokaryotic and eukaryotic cells. _Proc. R. Soc. Lond. B_ 266,

1571–1577 (1999). Article  CAS  Google Scholar  * Drew, B. & Leeuwenburgh, C. Method for measuring ATP production in isolated mitochondria: ATP production in brain and liver mitochondria

of Fischer-344 rats with age and caloric restriction. _Am. J. Physiol._ 285, R1259–R1267 (2003). CAS  Google Scholar  Download references 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 * School of Materials Science

and Engineering, Shanghai Jiao Tong University, Shanghai, P. R. China Can Xu, Mei Li & Stephen Mann * Institute of Medical Robotics, Shanghai Jiao Tong University, Shanghai, P. R. China

Can Xu * Centre for Protolife Research and Centre for Organized Matter Chemistry, School of Chemistry, University of Bristol, Bristol, UK Mei Li & Stephen Mann * Univ. Bordeaux, CNRS,

Centre de Recherche Paul Pascal, Pessac, France Nicolas Martin * Max Planck-Bristol Centre for Minimal Biology, School of Chemistry, University of Bristol, Bristol, UK Stephen Mann *

Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai, P. R. China Stephen Mann Authors * Can Xu View author publications You can also search for this author

inPubMed Google Scholar * Mei Li View author publications You can also search for this author inPubMed Google Scholar * Nicolas Martin View author publications You can also search for this

author inPubMed Google Scholar * Stephen Mann View author publications You can also search for this author inPubMed Google Scholar 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. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION

_Nature Protocols_ thanks Yan Qiao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature

remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. KEY REFERENCE Xu, C. et al. _Nature_ 609, 1029–1037 (2022):

https://doi.org/10.1038/s41586-022-05223-w SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figs. 1–9. REPORTING SUMMARY SOURCE DATA SOURCE DATA FIG. 5 Statistical Source

Data. SOURCE DATA FIG. 6 Statistical Source Data. RIGHTS AND PERMISSIONS Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a

publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing

agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Xu, C., Li, M., Martin, N. _et al._ Construction of complex bacteriogenic protocells from living

material assembly. _Nat Protoc_ (2025). https://doi.org/10.1038/s41596-025-01148-6 Download citation * Received: 08 July 2024 * Accepted: 13 January 2025 * Published: 05 March 2025 * DOI:

https://doi.org/10.1038/s41596-025-01148-6 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not

currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative