Life by Assembly Line: A Century-Long Quest to Build Life-And Define It

Abstract

Synthetic biology increasingly pursues the construction of engineered biological systems, yet the field lacks operational categories for interpreting claims of “life creation”. Rather than asking whether life has already been created in the laboratory, this article argues that synthetic biology requires a pragmatic framework that distinguishes modification, reconstruction, assembly, and autonomous synthesis of biological systems. Building on a historical analysis of recurring life-creation claims throughout twentieth-century biology, we develop a taxonomy that situates contemporary synthetic biology and xenobiology along a continuum of increasing engineering depth. Current achievements-including genome rewriting, orthogonal translation systems, and expanded genetic codes-represent a deep reconstruction of living systems, but do not yet constitute fully autonomous synthetic life. To clarify these distinctions, we introduce several conceptual tools: the Pasteurian Wall separating living from non-living systems, the Genetic Firewall as a biosafety principle for engineered organisms, and an Expanded Chemoton framework that provides an engineering-oriented operational definition of life based on metabolic autonomy, informational closure, and evolvability. Together, these elements allow experimental systems to be positioned along a functional continuum from sophisticated biochemical artifacts to genuinely alternative living systems. By replacing metaphor-driven narratives with operational categories, this framework aims to strengthen conceptual clarity, experimental comparability, and governance of emerging synthetic life technologies.

Keywords

Biosafety / Expanded chemoton / Genetic firewall / Life synthesis / Pasteurian wall / Operational definition / Synthetic biology / Xenobiology

References

[1] Budisa N. Life at the Speed of Light. From the Double Helix to the Dawn of Digital Life. Von J. Craig Venter. Angew. Chem. 2014, 126, 9575-9576. DOI: 10.1002/ange.201405385

[2] Endy D. Foundations for engineering biology. Nature 2005, 438, 449-453. DOI: 10.1038/nature04342

[3] Heinemann M, Panke S. Synthetic biology-Putting engineering into biology. Bioinformatics 2006, 22, 2790-2799. DOI: 10.1093/bioinformatics/btl469

[4] Shetty RP, Endy D, Knight TF . Engineering BioBrick vectors from BioBrick parts. J. Biol. Eng. 2008, 2, 5. DOI: 10.1186/1754-1611-2-5

[5] Smolke CD . Building outside of the box: iGEM and the BioBricks Foundation. Nat. Biotechnol. 2009, 27, 1099-1102. DOI: 10.1038/nbt1209-1099

[6] Purnick PEM, Weiss R . The second wave of synthetic biology: From modules to systems. Nat. Rev. Mol. Cell Biol. 2009, 10, 410-422. DOI: 10.1038/nrm2698

[7] Schrödinger E. What Is Life? The Physical Aspect of the Living Cell ; Rare Treasure Editions: Online, 2025.

[8] Luu HTL, Karbalaei‐Heidari HR, Budisa N . Essential Logic and Facts Behind the Expansion of the Genetic Code: A Critical Assessment. ChemCatChem 2025, 17, e00809. DOI: 10.1002/cctc.202500809

[9] Lillie RS. Living Systems and Non-Living Systems. Philos. Sci. 1942, 9, 307-322. DOI: 10.1086/286778

[10] Murase A. The Homunculus and the ParacelsianLiber de imaginibus . Ambix 2020, 67, 47-61. DOI: 10.1080/00026980.2020.1720359

[11] Kalra B, Baruah M, Kalra S . The Mahabharata and reproductive endocrinology. Indian J. Endocr. Metab. 2016, 20, 404-407. DOI: 10.4103/2230-8210.180004

[12] Ablondi F. Automata, Living and Non-Living: Descartes’ Mechanical Biology and His Criteria for Life. Biol. Philos. 1998, 13, 179-186. DOI: 10.1023/A:1006502002257

[13] Schmidt M, Pei L, Budisa N . Xenobiology: State-of-the-Art, Ethics, and Philosophy of New-to-Nature Organisms. In Synthetic Biology-Metabolic Engineering ; Springer: Cham, Switzerland, 2017; pp. 301-315. DOI: 10.1007/10_2016_14

[14] Zhang Y, Chen Y, Liao B . Analysis of the intrinsic value of life in the context of synthetic biology. Front. Bioeng. Biotechnol. 2025, 13, 1536403. DOI: 10.3389/fbioe.2025.1536403

[15] Kinne-Saffran E, Kinne RKH . Vitalism and Synthesis of Urea. Am. J. Nephrol. 1999, 19, 290-294. DOI: 10.1159/000013463

[16] Buchholz K, Bornscheuer UT . Enzyme Technology: History and Current Trends. In Applied Bioengineering: Innovations and Future Directions ; Wiley: Hoboken, NJ, USA, 2017; pp. 11-46. DOI: 10.1002/9783527800599.ch2

[17] Agostini F, Völler J, Koksch B, Acevedo‐Rocha CG, Kubyshkin V, Budisa N . Biocatalysis with Unnatural Amino Acids: Enzymology Meets Xenobiology. Angew. Chem. Int. Ed. 2017, 56, 9680-9703. DOI: 10.1002/anie.201610129

[18] Trifonov EN . Vocabulary of Definitions of Life Suggests a Definition. J. Biomol. Struct. Dyn. 2011, 29, 259-266. DOI: 10.1080/073911011010524992

[19] Budisa N. The genetic code as an operational a priori: Xenobiology and the rewriting of life’s grammar. Front. Synth. Biol. 2026, 4, e1760385. DOI: 10.3389/fsybi.2026.1760385

[20] Kriebisch CME, Bantysh O, Baranda Pellejero L, Belluati A, Bertosin E, Dai K, et al. A roadmap toward the synthesis of life. Chem 2025, 11, 102399. DOI: 10.1016/j.chempr.2024.102399

[21] Kuhn TS. The Structure of Scientific Revolutions ; University of Chicago Press: Chicago, IL, USA, 1997.

[22] Lane N. Transformer: The Deep Chemistry of Life and Death ; Profile Books: London, UK, 2022.

[23] Schummer J. ‘Are You Playing God?’: Synthetic Biology and the Chemical Ambition to Create Artificial Life. HYLE-Int. J. Philos. Chem. 2016, 22, 149-172. DOI: 10.5840/hyle20162218

[24] Wilkins J. Scientists Say They’ve Created a New Form of Life More Perfect Than the One Nature Made ; Futurism: New York, NY, USA, 2025.

[25] Wang K. Welcome to Post-Darwinism: AI Fires Silver Bullet for Creating New Life ; The Times: London, UK, 2026.

[26] King SH, Driscoll CL, Li DB, Guo D, Merchant AT, Brixi G, et al. Generative design of novel bacteriophages with genome language models. BioRxiv 2025, 2025-09. DOI: 10.1101/2025.09.12.675911

[27] Budisa N, Levin DB . Historical paradigm shifts in defining life: From spontaneous generation and vitalism to the Pasteurian Wall and the quest for artificial creation. Front. Synth. Biol. 2025, 3, 1692648. DOI: 10.3389/fsybi.2025.1692648

[28] Kubyshkin V, Budisa N . Synthetic alienation of microbial organisms by using genetic code engineering: Why and how? Biotechnol. J. 2017, 12, 1600097. DOI: 10.1002/biot.201600097

[29] White DH. A theory for the origin of a self-replicating chemical system. I: Natural selection of the autogen from short, random oligomers. J. Mol. Evol. 1980, 16, 121-147. DOI: 10.1007/BF01731582

[30] National Research Council. The Limits of Organic Life in Planetary Systems ; National Academies Press: Washington, DC, USA, 2007.

[31] Smith E, Morowitz HJ . The Origin and Nature of Life on Earth: The Emergence of the Fourth Geosphere ; Cambridge University Press: Cambridge, UK, 2016.

[32] Funk M, Steizinger J, Falkner D, Eichinger T. From Buzz to Burst-Critical Remarks on the Term ‘Life’ and Its Ethical Implications in Synthetic Biology. Nanoethics 2019, 13, 173-198. DOI: 10.1007/s11569-019-00361-4

[33] Bensaude Vincent B. Discipline-building in synthetic biology. Stud. Hist. Philos. Sci. Part C Stud. Hist. Philos. Biol. Biomed. Sci. 2013, 44, 122-129. DOI: 10.1016/j.shpsc.2013.03.007

[34] Heams T. Randomness in biology. Math. Struct. Comp. Sci. 2014, 24, e240308. DOI: 10.1017/S096012951200076X

[35] Otto S. An Approach to the De Novo Synthesis of Life . Acc. Chem. Res. 2022, 55, 145-155. DOI: 10.1021/acs.accounts.1c00534

[36] Schwille P, Spatz J, Landfester K, Bodenschatz E, Herminghaus S, Sourjik V, et al. MaxSynBio: Avenues Towards Creating Cells from the Bottom Up. Angew. Chem. Int. Ed. 2018, 57, 13382-13392. DOI: 10.1002/anie.201802288

[37] Luisi PL . Toward the engineering of minimal living cells. Anat. Rec. 2002, 268, 208-214. DOI: 10.1002/ar.10155

[38] Acevedo‐Rocha CG, Budisa N . On the Road towards Chemically Modified Organisms Endowed with a Genetic Firewall. Angew. Chem. Int. Ed. 2011, 50, 6960-6962. DOI: 10.1002/anie.201103010

[39] Karbalaei-Heidari HR, Budisa N . Advanced and Safe Synthetic Microbial Chassis with Orthogonal Translation System Integration. ACS Synth. Biol. 2024, 13, 2992-3002. DOI: 10.1021/acssynbio.4c00437

[40] Diwo C, Budisa N . Alternative Biochemistries for Alien Life: Basic Concepts and Requirements for the Design of a Robust Biocontainment System in Genetic Isolation. Genes 2018, 10, 17. DOI: 10.3390/genes10010017

[41] Haigh E. The roots of the vitalism of Xavier Bichat. Bull. Hist. Med. 1975, 49, 72-86. Available online: https://www.jstor.org/stable/44450204 (accessed on 26 February 2026).

[42] Monti M, Redi C. The egg. The inside story of a cell. Mol. Reprod. Dev. 2013, 80, 691-697. DOI: 10.1002/mrd.22196

[43] Ivanov I, Castellanos SL, Balasbas S, Otrin L, Marušič N, Vidaković-Koch T, et al. Bottom-Up Synthesis of Artificial Cells: Recent Highlights and Future Challenges. Annu. Rev. Chem. Biomol. Eng. 2021, 12, 287-308. DOI: 10.1146/annurev-chembioeng-092220-085918

[44] Liu Y, Sumpter DJT . Mathematical modeling reveals spontaneous emergence of self-replication in chemical reaction systems. J. Biol. Chem. 2018, 293, 18854-18863. DOI: 10.1074/jbc.RA118.003795

[45] Marliere P. The farther, the safer: A manifesto for securely navigating synthetic species away from the old living world. Syst. Synth. Biol. 2009, 3, 77-84. DOI: 10.1007/s11693-009-9040-9

[46] Vernadsky VI. The Biosphere ; Springer Science & Business Media: Berlin/Heidelberg, Germany, 1998.

[47] Handal-Marquez P, Anupama A, Pezo V, Marlière P, Herdewijn P, Pinheiro VB . Beneath the XNA world: Tools and targets to build novel biology. Curr. Opin. Syst. Biol. 2020, 24, 142-152. DOI: 10.1016/j.coisb.2020.10.013

[48] Schwille P. Bottom-Up Synthetic Biology: Engineering in a Tinkerer’s World. Science 2011, 333, 1252-1254. DOI: 10.1126/science.1211701

[49] Schmidt M. Xenobiology: A new form of life as the ultimate biosafety tool. BioEssays 2010, 32, 322-331. DOI: 10.1002/bies.200900147

[50] Danielli JF . The Artificial Synthesis of New Life Forms in Relation to Social and Industrial Evolution. In The Future of Man; Elsevier: Amsterdam, The Netherlands, 1972; pp. 95-112.

[51] Mandell DJ, Lajoie MJ, Mee MT, Takeuchi R, Kuznetsov G, Norville JE, et al. Biocontainment of genetically modified organisms by synthetic protein design. Nature 2015, 518, 55-60. DOI: 10.1038/nature14121

[52] Cowie DB, Cohen GN . Biosynthesis by Escherichia coli of active altered proteins containing selenium instead of sulfur. Biochim. Et. Biophys. Acta 1957, 26, 252-261. DOI: 10.1016/0006-3002(57)90003-3

[53] Pezo V, Metzgar D, Hendrickson TL, Waas WF, Hazebrouck S, Döring V, et al. Artificially ambiguous genetic code confers growth yield advantage. Proc. Natl. Acad. Sci. USA 2004, 101, 8593-8597. DOI: 10.1073/pnas.0402893101

[54] Schwander T, Schada von Borzyskowski L, Burgener S, Cortina NS, Erb TJ . A synthetic pathway for the fixation of carbon dioxide in vitro . Science 2016, 354, 900-904. DOI: 10.1126/science.aah5237

[55] Lovley DR, Nevin KP . A shift in the current: New applications and concepts for microbe-electrode electron exchange. Curr. Opin. Biotechnol. 2011, 22, 441-448. DOI: 10.1016/j.copbio.2011.01.009

[56] Danino T, Mondragón-Palomino O, Tsimring L, Hasty J . A synchronized quorum of genetic clocks. Nature 2010, 463, 326-330. DOI: 10.1038/nature08753

[57] Hyman AA, Weber CA, Jülicher F . Liquid-Liquid Phase Separation in Biology. Annu. Rev. Cell Dev. Biol. 2014, 30, 39-58. DOI: 10.1146/annurev-cellbio-100913-013325

[58] Gellman SH . Foldamers: A Manifesto. Acc. Chem. Res. 1998, 31, 173-180. DOI: 10.1021/ar960298r

[59] Agostini F, Sinn L, Petras D, Schipp CJ, Kubyshkin V, Berger AA, et al. Multiomics Analysis Provides Insight into the Laboratory Evolution of Escherichia coli toward the Metabolic Usage of Fluorinated Indoles. ACS Cent. Sci. 2021, 7, 81-92. DOI: 10.1021/acscentsci.0c00679

[60] Hoesl MG, Oehm S, Durkin P, Darmon E, Peil L, Aerni H, et al. Chemical Evolution of a Bacterial Proteome. Angew. Chem. Int. Ed. 2015, 54, 10030-10034. DOI: 10.1002/anie.201502868

[61] Maturana HR, Varela FJ . Autopoiesis and Cognition: The Realization of the Living ; Springer Science & Business Media: Berlin/Heidelberg, Germany, 2012.

[62] Schulze-Makuch D, Irwin LN . Life in the Universe ; Springer: Berlin/Heidelberg, Germany, 2004.

[63] Gánti T. The Principles of Life ; Oxford University Press: Oxford, UK, 2003.

[64] Ma HW, Zeng AP . The connectivity structure, giant strong component and centrality of metabolic networks. Bioinformatics 2003, 19, 1423-1430. DOI: 10.1093/bioinformatics/btg177

[65] Bölker M, Engelhard M, Budisa N . Synthetic Biology: Diverse Layers of Live. In Synthetic Biology Analysed: Tools for Discussion and Evaluation ; Springer: Berlin/Heidelberg, Germany, 2016; pp. 27-50.

[66] Marlière P, Patrouix J, Döring V, Herdewijn P, Tricot S, Cruveiller S, et al. Chemical Evolution of a Bacterium’s Genome. Angew. Chem. Int. Ed. 2011, 50, 7109-7114. DOI: 10.1002/anie.201100535

[67] Kambouris ME. Synthetic Biology as a Major Biothreat Enhancer. In Preparing for the Meta-Biothreat: Disruptive Biosecurity ; Springer: Cham, Switzerland, 2026; pp. 69-86.

[68] Pauly PJ . Controlling Life: Jacques Loeb and the Engineering Ideal in Biology ; Oxford University Press: Oxford, UK, 1987.

[69] Loeb J. On Artificial Parthenogenesis in Sea Urchins. Science 1900, 11, 612-614. DOI: 10.1126/science.11.277.612

[70] Peretó J. Erasing Borders: A Brief Chronicle of Early Synthetic Biology. J. Mol. Evol. 2016, 83, 176-183. DOI: 10.1007/s00239-016-9774-4

[71] Marshall M. A Radical New Theory Rewrites the Story of How Life on Earth Began ; NewScientist: London, UK, 2020.

[72] Joyce GF, Orgel LE . Prospects for Understanding the Origin of the RNA World. In The RNA World, 2nd ed.; Gesteland RF, Cech TR, Atkins JF, Eds.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, USA, 1999; pp. 49-77.

[73] Sutherland JD . The Origin of Life-Out of the Blue. Angew. Chem. Int. Ed. 2016, 55, 104-121. DOI: 10.1002/anie.201506585

[74] Leduc S. La biologie synthétique: A ; Poinat: Marseille, France, 1912.

[75] Thompson DAW. On Growth and Form ; Beyond Books Hub: Hong Kong, China, 2025.

[76] Cintas P. Chasing Synthetic Life: A Tale of Forms, Chemical Fossils, and Biomorphs. Angew. Chem. 2020, 132, 7364-7372. DOI: 10.1002/ange.201915853

[77] Rasmussen S, Chen L, Nilsson M, Abe S . Bridging Nonliving and Living Matter. Artif. Life 2003, 9, 269-316. DOI: 10.1162/106454603322392479

[78] Cardoso SSS, Cartwright JHE, Čejková J, Cronin L, De Wit A, Giannerini S, et al. Chemobrionics: From Self-Assembled Material Architectures to the Origin of Life. Artif. Life 2020, 26, 315-326. DOI: 10.1162/artl_a_00323

[79] Sharma A, Czégel D, Lachmann M, Kempes CP, Walker SI, Cronin L. Assembly theory explains and quantifies selection and evolution. Nature 2023, 622, 321-328. DOI: 10.1038/s41586-023-06600-9

[80] Luisi PL . The Emergence of Life: From Chemical Origins to Synthetic Biology ; Cambridge University Press: Cambridge, UK, 2016.

[81] Deamer D, Weber AL . Bioenergetics and Life’s Origins. Cold Spring Harb. Perspect. Biol. 2010, 2, a004929. DOI: 10.1101/cshperspect.a004929

[82] Kauffman SA . Approaches to the Origin of Life on Earth. Life 2011, 1, 34-48. DOI: 10.3390/life1010034

[83] Stein WD. James Frederic Danielli, 13 November 1911-22 April 1984 ; The Royal Society: London, UK, 1986

[84] Danielli JF. Genetic Engineering and Life Synthesis: An Introduction to the Review by R. Widdus and C. Ault. In International Review of Cytology; Elsevier: Amsterdam, The Netherlands, 1974; pp. 1-5.

[85] Robertson JD. The unit membrane and the Danielli-Davson model. In Intracellular Transport; Elsevier: Amsterdam, The Netherlands, 1966; pp. 1-31.

[86] Danielli JF . Artificial Synthesis of New Life Forms. Bull. At. Sci. 1972, 28, 20-24. DOI: 10.1080/00963402.1972.11457993

[87] Loose M, Kruse K, Schwille P . Protein Self-Organization: Lessons from the Min System. Annu. Rev. Biophys. 2011, 40, 315-336. DOI: 10.1146/annurev-biophys-042910-155332

[88] Holmes B. Alive! The Race to Create Life from Scratch ; NewScientist: London, UK, 2005.

[89] Stent GS . That Was the Molecular Biology That Was. Science 1968, 160, 390-395. DOI: 10.1126/science.160.3826.390

[90] Vendeville A, Larivière D, Fourmentin E . An inventory of the bacterial macromolecular components and their spatial organization. FEMS Microbiol. Rev. 2011, 35, 395-414. DOI: 10.1111/j.1574-6976.2010.00254.x

[91] Binder PM, Danchin A . Life’s demons: Information and order in biology. EMBO Rep. 2011, 12, 495-499. DOI: 10.1038/embor.2011.83

[92] Fischer E. Syntheses in the Purine and Sugar Group ; Nobel Lecture: Stockholm, Sweden, 1902; p. 12.

[93] Lichtenthaler FW . 100 Years “Schlüssel‐Schloss‐Prinzip”: What Made Emil Fischer Use this Analogy? Angew. Chem. Int. Ed. Engl. 1995, 33, 2364-2374. DOI: 10.1002/anie.199423641

[94] Fischer E. Die Kaiser-Wilhelm-Institute und der Zusammenhang von organischer Chemie und Biologie. In Untersuchungen aus Verschiedenen Gebieten ; Springer: Berlin/Heidelberg, Germany, 1924; pp. 796-809.

[95] Fischer E. Faraday lecture. Synthetical chemistry in its relation to biology. J. Chem. Soc. Trans. 1907, 91, 1749. DOI: 10.1039/ct9079101749

[96] Loeb J. Das Leben: Vortrag gehalten auf dem Ersten Monisten-Kongresse zu Hamburg am 10. September 1911 ; A. Kröner: Stuttgart, Germany, 1911.

[97] Deichmann U. Chemistry and the Engineering of Life Around 1900: Research and Reflections by Jacques Loeb. Biol Theory 2009, 4, 323-332. DOI: 10.1162/BIOT_a_00001

[98] Schummer J. Das Gotteshandwerk. Die künstliche Herstellung von Leben im Labor ; Suhrkamp: Berlin, Germany, 2011; p. 111.

[99] Bundell S. Complex synthetic cells bring scientists closer to artificial cellular life. Nature 2022. DOI: 10.1038/d41586-022-02946-8

[100] Pinheiro VB, Taylor AI, Cozens C, Abramov M, Renders M, Zhang S, et al. Synthetic Genetic Polymers Capable of Heredity and Evolution. Science 2012, 336, 341-344. DOI: 10.1126/science.1217622

[101] Chaput JC, Egli M, Herdewijn P . The XNA alphabet. Nucleic Acids Res. 2025, 53, gkaf635. DOI: 10.1093/nar/gkaf635

[102] Romesberg FE. Creation, Optimization, and Use of Semi-Synthetic Organisms that Store and Retrieve Increased Genetic Information. J. Mol. Biol. 2022, 434, 167331. DOI: 10.1016/j.jmb.2021.167331

[103] Bain JD, Switzer C, Chamberlin R, Benner SA . Ribosome-mediated incorporation of a non-standard amino acid into a peptide through expansion of the genetic code. Nature 1992, 356, 537-539. DOI: 10.1038/356537a0

[104] Koch NG, Budisa N . Evolution of Pyrrolysyl-tRNA Synthetase: From Methanogenesis to Genetic Code Expansion. Chem. Rev. 2024, 124, 9580-9608. DOI: 10.1021/acs.chemrev.4c00031

[105] Murgola E. tRNA, Suppression, and the Code. Annu. Rev. Genet. 1985, 19, 57-80. DOI: 10.1146/annurev.genet.19.1.57

[106] Baumann T, Hauf M, Richter F, Albers S, Möglich A, Ignatova Z, et al. Computational Aminoacyl-tRNA Synthetase Library Design for Photocaged Tyrosine. Int. J. Mol. Sci. 2019, 20, 2343. DOI: 10.3390/ijms20092343

[107] Kolber NS, Fattal R, Bratulic S, Carver GD, Badran AH . Orthogonal translation enables heterologous ribosome engineering inE. coli . Nat. Commun. 2021, 12, 599. DOI: 10.1038/s41467-020-20759-z

[108] Grosjean H, Westhof E. An integrated, structure- and energy-based view of the genetic code. Nucleic Acids Res. 2016, 44, 8020-8040. DOI: 10.1093/nar/gkw608

[109] Atkins JF, Gesteland RF, Atkins RFGJF . Recoding ; Springer: Berlin/Heidelberg, Germany, 2009.

[110] Kubyshkin V, Budisa N . Anticipating alien cells with alternative genetic codes: Away from the alanine world! Curr. Opin. Biotechnol. 2019, 60, 242-249. DOI: 10.1016/j.copbio.2019.05.006

[111] Miller SL, Schopf JW, Lazcano A . Oparin’s “Origin of Life”: Sixty Years Later. J. Mol. Evol. 1997, 44, 351-353. DOI: 10.1007/PL00006153

[112] Miller SL, Urey HC . Organic Compound Synthesis on the Primitive Earth. Science 1959, 130, 245-251. DOI: 10.1126/science.130.3370.245

[113] Schrum JP, Zhu TF, Szostak JW . The Origins of Cellular Life. Cold Spring Harb. Perspect. Biol. 2010, 2, a002212. DOI: 10.1101/cshperspect.a002212

[114] Fry I. The origins of research into the origins of life. Endeavour 2006, 30, 24-28. DOI: 10.1016/j.endeavour.2005.12.002

[115] Berg P. Asilomar and recombinant DNA. (News). Sci. 2002, 16, 19-20. Available online: https://go.gale.com/ps/i.do?id=GALE%7CA84542712&sid=googleScholar&v=2.1&it=r&linkaccess=abs&issn=08903670&p=AONE&sw=w (accessed on 24 February 2026).

[116] Gepts P. A Comparison between Crop Domestication, Classical Plant Breeding, and Genetic Engineering. Crop Sci. 2002, 42, 1780-1790. DOI: 10.2135/cropsci2002.1780

[117] Monod J. On Chance and Necessity. In Studies in the Philosophy of Biology: Reduction and Related Problems ; Springer: Berlin/Heidelberg, Germany, 1974; pp. 357-375.

[118] Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang RY, Algire MA, et al. Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome. Science 2010, 329, 52-56. DOI: 10.1126/science.1190719

[119] Venter JC . Life at the Speed of Light: From the Double Helix to the Dawn of Digital Life ; Penguin: London, UK, 2013.

[120] Brown DM . A Brief History of Oligonucleotide Synthesis. In Protocols for Oligonucleotides and Analogs: Synthesis and Properties ; Springer: Berlin/Heidelberg, Germany, 2003; pp. 1-18.

[121] Lartigue C, Glass JI, Alperovich N, Pieper R, Parmar PP, Hutchison CA, et al. Genome Transplantation in Bacteria: Changing One Species to Another. Science 2007, 317, 632-638. DOI: 10.1126/science.1144622

[122] Macrae F. Scientist accused of playing God after creating artificial life by making designer microbe from scratch-but could it wipe out humanity. Dly. Mail. 2010, 3. Available online: http://www.dailymail.co.uk/sciencetech/article-1279988/Artificial-life-created-Craig-Venter-wipe-humanity.htm (accessed on 24 February 2026).

[123] Sullivan W. Bauffalo Scientists Report Synthesis of Living Cell ; The New York Times: New York, NY, USA, 1970.

[124] Church GM, Regis E. Regenesis: How Synthetic Biology Will Reinvent Nature and Ourselves ; Basic Books: New York, NY, USA, 2014.

[125] Bohannon J. The Life Hacker. Science 2011, 333, 1236-1237. DOI: 10.1126/science.333.6047.1236

[126] Boldt J, Müller O . Newtons of the leaves of grass. Nat. Biotechnol. 2008, 26, 387-389. DOI: 10.1038/nbt0408-387

[127] Bedau MA, Triant M . Social and Ethical Implications of Creating Artificial Cells. In Ethics and Emerging Technologies ; Springer: Berlin/Heidelberg, Germany, 2014; pp. 562-574.

[128] Feynman R. What I cannot create, I do not understand. Note on a Caltech blackboard. 1988. Available online: https://library.caltech.edu/archives/home (accessed on 24 February 2026).

[129] Miner RC . Verum-factum and Practical Wisdom in the Early Writings of Giambattista Vico. J. Hist. Ideas 1998, 59, 53-73. DOI: 10.1353/jhi.1998.0006

[130] Crupi V, Fitelson B, Tentori K . Probability, confirmation, and the conjunction fallacy. Think. Reason. 2008, 14, 182-199. DOI: 10.1080/13546780701643406

[131] Danchin A. Bacteria as computers making computers. FEMS Microbiol. Rev. 2009, 33, 3-26. DOI: 10.1111/j.1574-6976.2008.00137.x

[132] Agapakis CM, Silver PA . Synthetic biology: Exploring and exploiting genetic modularity through the design of novel biological networks. Mol. BioSystems 2009, 5, 704-713. DOI: 10.1039/B901484E

[133] Barrett HC, Kurzban R . Modularity in cognition: Framing the debate. Psychol. Rev. 2006, 113, 628-647. DOI: 10.1037/0033-295X.113.3.628

[134] Hofstra B, Kulkarni VV, Munoz-Najar Galvez S, He B, Jurafsky D, McFarland DA . The Diversity-Innovation Paradox in Science. Proc. Natl. Acad. Sci. USA 2020, 117, 9284-9291. DOI: 10.1073/pnas.1915378117

[135] Prabhu K. Use of GMOs under containment, confined and limited field trials and post-release monitoring of GMOs. In Biosafety of Genetically Modified Organisms: Basic Concepts, Methods and Issues; Chowdhury MKA, Hoque MI, Sonnino A, Eds.; Food and Agriculture Organization of the United Nations: Rome, Italy, 2009; pp. 157-220.

[136] Hickman T. Problems for proportionality. New Zealand Law. Rev. 2010, 2010, 303-326. DOI: 10.3316/agispt.20111111

[137] McLeod C, Nerlich B . Synthetic biology, metaphors and responsibility. Life Sci. Soc. Policy 2017, 13, 13. DOI: 10.1186/s40504-017-0061-y

[138] Szostak JW . Attempts to Define Life Do Not Help to Understand the Origin of Life. J. Biomol. Struct. Dyn. 2012, 29, 599-600. DOI: 10.1080/073911012010524998

[139] Gayon J. Defining Life: Synthesis and Conclusions. Orig. Life Evol. Biosph. 2010, 40, 231-244. DOI: 10.1007/s11084-010-9204-3

[140] Cleland C, Chyba C. Does ‘life’ have a definition? In The Nature of Life: Classical and Contemporary Perspectives from Philosophy and Science; Bedau MA, Cleland C E, Eds.; Cambridge University Press: Cambridge, UK, 2010; pp. 326-339.

[141] Laketic D, Tufte G . Autonomous adaptation inspired by the model of a minimal living system provided by chemoton theory. Nano Commun. Netw. 2011, 2, 125-140. DOI: 10.1016/j.nancom.2011.05.003

[142] Cornish-Bowden A . Tibor Gánti and Robert Rosen: Contrasting approaches to the same problem. J. Theor. Biol. 2015, 381, 6-10. DOI: 10.1016/j.jtbi.2015.05.015

[143] Popper KR . Science as falsification. Conjectures Refutations 1963, 1, 33-39. Available online: https://www.stephenhicks.org/wp-content/uploads/2018/09/PopperK-Science-as-Falsification.pdf (accessed on 24 February 2026).

[144] Joyce GF. The RNA World: Life Before DNA and Protein ; NTRS: Chicago, IL, USA, 1993.

[145] Benner SA . Defining Life. Astrobiology 2010, 10, 1021-1030. DOI: 10.1089/ast.2010.0524

[146] Popa R. Between Necessity and Probability: Searching for the Definition and Origin of Life ; Springer Science & Business Media: Berlin/Heidelberg, Germany, 2004.

[147] Joyce GF. The antiquity of RNA-based evolution. Nature 2002, 418, 214-221. DOI: 10.1038/418214a

[148] Bedau MA . Four Puzzles About Life. Artif. Life 1998, 4, 125-140. DOI: 10.1162/106454698568486

[149] de Duve C. Life Evolving: Molecules, Mind, and Meaning ; Oxford University Press: Oxford, UK, 2002.

[150] Sagan D, Whiteside JH . Gradient Reduction Theory: Thermodynamics and the Purpose of Life. In Scientists Debate Gaia: The Next Century; The MIT Press: Cambridge, MA, USA, 2004; pp. 173-186.

[151] Schulze-Makuch D, Irwin LN, Guan H . Search parameters for the remote detection of extraterrestrial life. Planet. Space Sci. 2002, 50, 675-683. DOI: 10.1016/S0032-0633(01)00121-0

[152] Schneider ED, Sagan D. Into the Cool: Energy Flow, Thermodynamics, and Life ; University of Chicago Press: Chicago, IL, USA, 2005.

[153] Budisa N, Schulze-Makuch D . Supercritical Carbon Dioxide and Its Potential as a Life-Sustaining Solvent in a Planetary Environment. Life 2014, 4, 331-340. DOI: 10.3390/life4030331

[154] Budisa N, Kubyshkin V, Schulze-Makuch D . Fluorine-Rich Planetary Environments as Possible Habitats for Life. Life 2014, 4, 374-385. DOI: 10.3390/life4030374

[155] Steizinger J. Engineers of Life? A Critical Examination of the Concept of Life in the Debate on Synthetic Biology. In Ambivalences of Creating Life: Societal and Philosophical Dimensions of Synthetic Biology; Springer: Berlin/Heidelberg, Germany, 2016; pp. 275-292.

[156] Noble D. Editorial: Charles Darwin, Jean-Baptiste Lamarck, and 21st century arguments on the fundamentals of biology. Prog. Biophys. Mol. Biol. 2020, 153, 1-4. DOI: 10.1016/j.pbiomolbio.2020.02.005

[157] Morowitz H, Smith E . Energy flow and the organization of life. Complexity 2007, 13, 51-59. DOI: 10.1002/cplx.20191

[158] Griesemer J, Szathmáry E . Gánti’s Chemoton Model and Life Criteria. Protocells 2008, 481-512. DOI: 10.7551/mitpress/9780262182683.003.0022

[159] Von Neumann J, Burks AW . Theory of Self-Reproducing Automata. 1966. Available online: http://www.cba.mit.edu/events/03.11.ASE/docs/VonNeumann.pdf (accessed on 24 February 2026).

[160] Barbieri M. The Organic Codes: An Introduction to Semantic Biology ; Cambridge University Press: Cambridge, UK, 2002.

[161] Ruiz-Mirazo K, Peretó J, Moreno A . Defining Life or Bringing Biology to Life. Orig. Life Evol. Biosph. 2010, 40, 203-213. DOI: 10.1007/s11084-010-9201-6

[162] Szathmáry E, Smith JM . The Major Transitions in Evolution ; WH Freeman Spektrum Oxford: Oxford, UK, 1995.

[163] Cornish-Bowden A, Cardenas ML . Bringing Chemistry to Life: What Does It Mean to Be Alive? 2006. Available online: https://www.beilstein-institut.de/download/661/cornish.pdf (accessed on 24 February 2026).

[164] Pace NR. The universal nature of biochemistry. Proc. Natl. Acad. Sci. USA 2001, 98, 805-808. DOI: 10.1073/pnas.98.3.805

[165] Bedau M, Church G, Rasmussen S, Caplan A, Benner S, Fussenegger M, et al. Life after the synthetic cell. Nature 2010, 465, 422-424. DOI: 10.1038/465422a

[166] Spitzer J, Pielak GJ, Poolman B . Emergence of life: Physical chemistry changes the paradigm. Biol. Direct 2015, 10, 33. DOI: 10.1186/s13062-015-0060-y

[167] Bich L, Green S . Is defining life pointless? Operational definitions at the frontiers of biology. Synthese 2018, 195, 3919-3946. DOI: 10.1007/s11229-017-1397-9

[168] Ruiz-Mirazo K, Moreno A . Autonomy in evolution: From minimal to complex life. Synthese 2012, 185, 21-52. DOI: 10.1007/s11229-011-9874-z

[169] Vitas M, Dobovišek A . Towards a General Definition of Life. Orig. Life Evol. Biosph. 2019, 49, 77-88. DOI: 10.1007/s11084-019-09578-5

[170] Koskinen R. Could We Really Be Made of Swiss Cheese? Xenobiology as an Engineering Epistemology for Biological Realization. ChemBioChem 2020, 21, 2591-2594. DOI: 10.1002/cbic.202000063

[171] Blackiston D, Lederer E, Kriegman S, Garnier S, Bongard J, Levin M . A cellular platform for the development of synthetic living machines. Sci. Robot. 2021, 6, eabf1571. DOI: 10.1126/scirobotics.abf1571

[172] Pols T, Sikkema HR, Gaastra BF, Frallicciardi J, Śmigiel WM, Singh S, et al. A synthetic metabolic network for physicochemical homeostasis. Nat. Commun. 2019, 10, 4239. DOI: 10.1038/s41467-019-12287-2

[173] Gaut NJ, Deich C, Cash B, Hoog T, Engelhart AE, Adamala KP . A Chemically Defined Synthetic Cell Capable of Growth And Replication. BioRxiv 2026. DOI: 10.64898/2026.07.01.735724

[174] Wiener N. Cybernetics or Control and Communication in the Animal and the Machine ; MIT Press: Cambridge, MA, USA, 2019. DOI: 10.7551/mitpress/11810.001.0001

[175] Keller EF. The Century of the Gene ; Harvard University Press: Cambridge, MA, USA, 2000. DOI: 10.4159/9780674039438

[176] Deacon TW . Incomplete Nature: How Mind Emerged from Matter ; W. W. Norton & Company: New York, NY, USA, 2011.

[177] Cleland CE . Life without definitions. Synthese 2012, 185, 125-144. DOI: 10.1007/s11229-011-9879-7

[178] McKay CP . What Is Life-And How Do We Search for It in Other Worlds? PLoS Biol. 2004, 2, e302. DOI: 10.1371/journal.pbio.0020302

[179] Godfrey-Smith P. Darwinian Populations and Natural Selection ; Oxford University Press: Oxford, UK, 2009. DOI: 10.1093/acprof:osobl/9780199552047.001.0001

[180] Pross A. What Is Life? How Chemistry Becomes Biology ; Oxford University Press: Oxford, UK, 2016.

[181] Mayr E. The Growth of Biological Thought: Diversity, Evolution, and Inheritance ; Harvard University Press: Cambridge, MA, USA, 1982.

[182] Woese CR . On the evolution of cells. Proc. Natl. Acad. Sci. USA 2002, 99, 8742-8747. DOI: 10.1073/pnas.132266999

[183] Dobzhansky T. Genetics and the Origin of Species ; Columbia University Press: New York, NY, USA, 1982.

[184] Lynch M, Walsh B . The Origins of Genome Architecture ; Sinauer Associates: Sunderland, MA, USA, 2007.

[185] Jewett MC, Forster AC . Update on designing and building minimal cells. Curr. Opin. Biotechnol. 2010, 21, 697-703. DOI: 10.1016/j.copbio.2010.06.008

[186] Smith E, Morowitz HJ . Universality in intermediary metabolism. Proc. Natl. Acad. Sci. USA 2004, 101, 13168-13173. DOI: 10.1073/pnas.0404922101

[187] Budisa N, Kubyshkin V, Schmidt M . Xenobiology: A Journey towards Parallel Life Forms. ChemBioChem 2020, 21, 2228-2231. DOI: 10.1002/cbic.202000141

[188] Liu Y, Di Z, Gerlee P . Ladderpath Approach: How Tinkering and Reuse Increase Complexity and Information. Entropy 2022, 24, 1082. DOI: 10.3390/e24081082

[189] y Arcas BA . What Is Life? Evolution as Computation ; MIT Press: Cambridge, MA, USA, 2025.

[190] Kempes CP, Krakauer DC . The Multiple Paths to Multiple Life. J. Mol. Evol. 2021, 89, 415-426. DOI: 10.1007/s00239-021-10016-2

[191] Kirschning A. Coenzymes and Their Role in the Evolution of Life. Angew. Chem. Int. Ed. 2021, 60, 6242-6269. DOI: 10.1002/anie.201914786

[192] Bitzenhofer NL, Kruse L, Thies S, Wynands B, Lechtenberg T, Rönitz J, et al. Towards robust Pseudomonas cell factories to harbour novel biosynthetic pathways. Essays Biochem. 2021, 65, 319-336. DOI: 10.1042/EBC20200173

[193] Sackmann E. Physical Basis of Self-Organization and Function of Membranes: Physics of Vesicles. InHandbook of Biological Physics. 1 ; Elsevier: Amsterdam, The Netherlands, 1995; pp. 213-304. DOI: 10.1016/S1383-8121(06)80022-9

[194] Schmidt M, Kubyshkin V . How To Quantify a Genetic Firewall? A Polarity‐Based Metric for Genetic Code Engineering. ChemBioChem 2021, 22, 1268-1284. DOI: 10.1002/cbic.202000758

[195] Hoesl MG, Larregola M, Cui H, Budisa N . Azatryptophans as tools to study polarity requirements for folding of green fluorescent protein. J. Pept. Sci. 2010, 16, 589-595. DOI: 10.1002/psc.1263

[196] Lavoisier AL . Traité élémentaire de chimie ; Chez Deterville: Paris, France, 1864.

[197] Bensaude-Vincent B. A view of the chemical revolution through contemporary textbooks: Lavoisier, Fourcroy and Chaptal. Brit. J. Hist. Sci. 1990, 23, 435-460. DOI: 10.1017/S0007087400028089

[198] Tolle I, Oehm S, Hoesl MG, Treiber-Kleinke C, Peil L, Bozukova M, et al. Evolving a mitigation of the stress response pathway to change the basic chemistry of life. Front. Synth. Biol. 2023, 1, 1248065. DOI: 10.3389/fsybi.2023.1248065

[199] Nelson DL, Lehninger AL, Cox MM . Lehninger Principles of Biochemistry ; Macmillan: New York, NY, USA, 2008.

[200] Fasman GD . Circular Dichroism and the Conformational Analysis of Biomolecules ; Springer Science & Business Media: Berlin/Heidelberg, Germany, 2013.

[201] Kubyshkin V, Budisa N . The Alanine World Model for the Development of the Amino Acid Repertoire in Protein Biosynthesis. Int. J. Mol. Sci. 2019, 20, 5507. DOI: 10.3390/ijms20215507

[202] Adamala KP, Agashe D, Belkaid Y, Bittencourt DMDC, Cai Y, Chang MW, et al. Confronting risks of mirror life. Science 2024, 386, 1351-1353. DOI: 10.1126/science.ads9158

[203] Li YY, Moradialvand M, Asbridge LA, Frazier KB, Tay FR, Makvandi P . Mirror Life: Bridging Chirality, Ethics, and the Foundations of Life Creation. Adv. Mater. 2025, 38, e10791. DOI: 10.1002/adma.202510791

[204] Liu S. Homochirality Originates from the Handedness of Helices. J. Phys. Chem. Lett. 2020, 11, 8690-8696. DOI: 10.1021/acs.jpclett.0c02144

[205] Neidle S, Sanderson M . Principles of Nucleic Acid Structure ; Academic Press: Cambridge, MA, USA, 2021.

[206] Song Y, Wang S, Xu Y . Mirror-Image RNA: A Right-Handed Z-Form RNA and Its Ligand Complex. Molecules 2024, 29, 4900. DOI: 10.3390/molecules29204900

[207] Leslie E. O. Prebiotic Chemistry and the Origin of the RNA World. Crit. Rev. Biochem. Mol. Biol. 2004, 39, 99-123. DOI: 10.1080/10409230490460765

[208] Achenbach J-P. Through the Mirror: Translation with D-Amino Acids ; Technische Universitaet Berlin: Berlin, Germany, 2015.

[209] Eschenmoser A. Chemical Etiology of Nucleic Acid Structure. Science 1999, 284, 2118-2124. DOI: 10.1126/science.284.5423.2118

[210] Schulze-Makuch D, Irwin LN . The prospect of alien life in exotic forms on other worlds. Naturwissenschaften 2006, 93, 155-172. DOI: 10.1007/s00114-005-0078-6

[211] Rampelotto PH . The search for life on other planets: Sulfur-based, silicon-based, ammonia-based life. J. Cosmol. 2010, 5, 818-827. Available online: http://plutao.sid.inpe.br/J8LNKAN8RW/37PGAH5?mirror=dpi.inpe.br/plutao@80/2008/08.19.15.01.21&metadatarepository= (accessed on 24 February 2026).

[212] Irwin LN, Schulze-Makuch D . The Astrobiology of Alien Worlds: Known and Unknown Forms of Life. Universe 2020, 6, 130. DOI: 10.3390/universe6090130

[213] Szathmáry E. Coding coenzyme handles: A hypothesis for the origin of the genetic code. Proc. Natl. Acad. Sci. USA 1993, 90, 9916-9920. DOI: 10.1073/pnas.90.21.9916

[214] Budisa N. Xenobiology, New-to-Nature Synthetic Cells and Genetic Firewall. Curr. Org. Chem. 2014, 18, 936-943. DOI: 10.2174/138527281808140616154301

[215] Smith AA, Wong EL, Donovan RC, Chapman BA, Harry R, Tirandazi P, et al. Using a GPT-5-driven autonomous lab to optimize the cost and titer of cell-free protein synthesis. bioRxiv 2026. DOI: 10.64898/2026.02.05.703998

[216] Budisa N. Parallele biologische Welt mit genetischer Firewall-Wahrheit oder Dichtung? In Blickpunkt: Leben Am Rande des Daseins; Herzog EM, Bauer H-C, Lametschwandtner A, Eds.; Books on Demand (BoD): Norderstedt, Germany, 2014; pp. 93-106.

[217] Toepfer G. Leben. In Historisches Wörterbuch der Biologie ; Springer: Berlin/Heidelberg, Germany, 2011; pp. 420-483. DOI: 10.1007/978-3-476-00455-0_24

[218] Aparicio De Narvaez A. ‘Accept No Limits’: Imaginaries of Life, Responsibility and Biosafety in Xenobiology ; University College London: London, UK, 2019.

[219] Revol F. Theology of Continuous Creation. Theol. Sci. 2021, 19, 287-299. DOI: 10.1080/14746700.2021.1944500

[220] Höijer B. Emotional anchoring and objectification in the media reporting on climate change. Public. Underst. Sci. 2010, 19, 717-731. DOI: 10.1177/0963662509348863

[221] Jasanoff S. Designs on Nature: Science and Democracy in Europe and the United States ; Princeton University Press: Princeton, NJ, USA, 2005. DOI: 10.1515/9781400837311

[222] Rai AK, Eisenberg RS . Bayh-Dole Reform and the Progress of Biomedicine. Law Contemp. Probs. 2003, 66, 289. DOI: 10.2139/ssrn.348343

[223] Contreras JL. Unpacking Open Source Bio. Ind. LJ 2025, 100, 1567. DOI: 10.2139/ssrn.5043247

[224] Sampat BN, Shadlen KC . The COVID-19 Innovation System. Health Aff. 2021, 40, 400-409. DOI: 10.1377/hlthaff.2020.02097

[225] Hoagland H. I. Man's Responsibility to His Future. Bull. At. Sci. 1972, 28, 13. DOI: 10.1080/00963402.1972.11457990

[226] Kinne D. The Balenciaga is flowering: A speculative investigation into biosynthetic luxury. Lux. Stud. Purs. Lux. J. 2025, 4, 31-49. DOI: 10.1386/ipol_00042_1

[227] Rose N. The Value of Life: Somatic Ethics and the Spirit of Biocapital. In The Right to Life and the Value of Life; Routledge: London, UK, 2016; pp. 101-116. DOI: 10.4324/9781315553474-9

[228] Stoate R. Gray Goo and You: The Ecophagy of Global Capital. In Criticism, Crisis, and Contemporary Narrative ; Routledge: London, UK, 2011; pp. 120-136.

[229] Dunne A, Raby F . Speculative Everything, with a New Preface by the Authors: Design, Fiction, and Social Dreaming ; MIT Press: Cambridge, MA, USA, 2024.

[230] Hurlbut JB. Remembering the future: Science, law, and the legacy of Asilomar. In Dreamscapes of Modernity: Sociotechnical Imaginaries and the Fabrication of Power; University of Chicago Press: Chicago, IL, USA, 2015; pp. 126-151. DOI: 10.7208/chicago/9780226276663.003.0006

[231] Rabinow P, Bennett G . Designing Human Practices: An Experiment with Synthetic Biology ; University of Chicago Press: Chicago, IL, USA, 2012. DOI: 10.7208/chicago/9780226703152.001.0001

[232] Budisa N. Commentary: Evolving a mitigation of the stress response pathway to change the basic chemistry of life. Front. Synth. Biol. 2024, 2, 1380879. DOI: 10.3389/fsybi.2024.1380879