Synthesizing Life in the Laboratory: Why is it not Happening?

One of the founders of modern biochemistry, Jacques Loeb wrote in 1912 … “nothing indicates, however, at present that the artificial production of living matter is beyond the possibilities of science… we must succeed in producing living matter artificially, or we must find the reasons why this is impossible.”[1][2] This was written prior to the crystallization of a single protein, the discovery of the citric acid cycle and much of metabolism, before understanding the structure of DNA and the concepts of molecular biology. For all these reasons, Dr. Loeb may be excused for his premature optimism regarding the synthesis of living matter. Indeed, more than a century later, the concept of laboratory abiogenesis is just beginning to surface, due to the emerging field of synthetic biology.

The ultimate goal of synthetic biology is to produce living matter in the laboratory, in-vitro abiogenesis.[3][4][5] Although there is a lack of consensus in the scientific community as to what constitutes “living matter,”[6][7][8][9][10] a very helpful compilation of nine invariant characteristics of all living structures has been published.[11] But for the purpose of this communication, the most helpful characterization of a living entity is given in the insightful review of this field:[12] “… by “living” we mean the capacity of autonomous self-sustainment in an out-of-equilibrium homeostatic state, with the additional possibility of growth-and-division, giving rise to a sort of minimal life cycle, and to evolution”.

Because it is generally thought that at the very heart of all living matter, the “essence of life” is a requisite constellation of nucleic acids and proteins, all current efforts in synthetic biology are directed toward achieving model cells containing these components. Quoting a prominent scientist on this subject: “…life itself can be seen as an emergent property: the molecules that constitute a living cell (DNA, proteins, polysaccharides, lipids, etc.) are not living. The quality “life” arises from the assembly of these non-living elements, duly arranged in space and time.”[13]

Underlying all efforts in synthetic biology is the fundamentally crucial assumption that it is possible to assemble living matter stepwise from a set of biomolecules. A corollary of this supposition is that, at least in principle, living matter may be disassembled reversibly and reassembled. While such is the nearly universal consensus within the scientific community, as of now, these assumptions have not been verified experimentally.

The conviction that living matter may be crafted from inert organic molecules may be traced to the postulates of I. O Oparin[14] and J. D. Haldane[15] regarding the origins of life. With the discoveries of S. Miler and H. Urey[16] this notion blossomed into the field of chemical evolution, which over the last three quarters of century produced a prodigious body of work[17] but little clarity as to how living matter could have come into existence in a primitive earth setting.

Synthetic biology is liberated from the considerable burden of doing biochemistry under primordial conditions. Now laboratories are free to use whatever means they have available to construct living matter! Indeed, there is considerable optimism that synthetic biology will finally accomplish the “holy grail” of biology, the production of an artificial living cell. Accordingly, a concluding remark of a relatively recent review article on the subject states: “The synthesis of a living artificial cell from components will open the door to many more adventurous lines of research…”[18] However, a more cautious reviewer of the subject states: “…it is important to note that minimal life has not yet been achieved in the laboratory. Does this mean that it is in principle not possible? I do not believe so, although as a scientist it is always good to have a bit of doubt (perhaps we missed something important in our theoretical analysis)”.[19]

This communication points to just such an oversight, the underestimation of the essential nature of the “out-of equilibrium” state of living matter.

All life processes, metabolism, growth, stimulus response and replication are driven by on-going chemical reactions. Every chemical reaction exists in one of two states, non-equilibrium and equilibrium. On-going chemical processes are always in states of non-equilibrium.

When a chemical reaction, aA + bB ⇌ cC + dD runs its course, equilibrium ensues, where the mass action ratio Γ=[C]cx[D]d/[A]ax[B]b becomes the equilibrium constant, Keq. At equilibrium, the change in free energy ∆F=0 and in this state the reaction cannot generate or absorb any energy.

During chemical reactions there is a net flux of matter from reactants to products or the reverse. However, at equilibrium the flux stops.

Moreover, the state of equilibrium resists change. As the Le Chatelier’s principle[20] states, if a chemical system at equilibrium experiences a change in concentration, temperature, volume, or partial pressure, then the equilibrium shifts to counteract the imposed change and a new equilibrium is established. Thus, according to this principle, any change from a state of non-equilibrium to equilibrium is irreversible.

Even though in living cells each reaction is pushed toward equilibrium by an enzyme (so as to forestall the possibility of slower, random non-biological chemical events), if any of the hundreds to thousands of chemical processes could actually reach equilibrium, an irreversible metabolic block would result. Multiple such equilibriums would kill the cell. However, in live cells there are no isolated reactions and the problem of equilibrium is avoided. Rather, chemical events are linked into pathways, so that the products of reactions do not accumulate, but immediately react with another substance.

The end products of metabolic pathways are either utilized immediately or they are secreted from the cell. Moreover, regulatory systems such as “feedback inhibition” help maintain homeostasis.

That the non-equilibrium steady states of all chemical reactions/pathways in live cells constitute an essence of life can be shown to be true by the simple experiment of briefly treating an aliquot of growing Escherichia coli culture with drops of toluene.[21] This procedure creates holes in the outer membrane of the bacterium, causing the dissolution of the proton gradient between the cytoplasm and the periplasm.[22][23] In turn, ATP synthesis halts and within seconds the reactions in the cell reach their equilibriums and the organism dies.

At this point, the dead cell contains most of its nucleic acids, proteins, lipids, polysaccharides and metabolites. Therefore, while the genetic material, RNA, enzymes, polysaccharides and lipids are all necessary components of a live cell; their mere presence is not sufficient for life. In live cells, superimposed on all of the necessary biopolymers is the steady state non-equilibrium dynamics of all chemical events.

Even if it would be possible to prevent internal degradation of its biopolymers, the dead bacterium would never come back to life just by continued incubation.

Current practitioners of synthetic biology, while recognizing the compulsory “out-of-equilibrium homeostatic state” of living matter, do not appear to appreciate the irreversibility <non-spontaneity?> of the state of equilibrium. Building artificial cells in a modular fashion will inevitably result in the onset of chemical equilibrium within each module. Once equilibrium is reached, the artificial cell, figuratively speaking, “runs into a brick wall”. It is no longer capable of growth or accomplish any net chemical process.  

No technology is known to achieve modular assembly of artificial cells while preserving the non-equilibrium status of each component reaction. While these considerations do not apply to polymerizations, such as RNA or DNA synthesis, as each incremental extension of the polymer is accompanied by the hydrolysis of a high-energy bond rendering these steps essentially irreversible, any other metabolic event is very much subject to termination due to reaching equilibrium. Until the construction of cell-like structures harboring metabolisms in homeostatic non-equilibrium states become reality, the most sophisticated efforts of synthetic biology will come to naught.

Therefore, more than a century later, our response to Jacques Loeb’s call for the synthesis of living matter is that we are not there yet. We need to find ways to generate steady state non-equilibrium conditions within the artificial cells. These technologies await inventions in the future.

George T. Javor, PhD


[1] Loeb, J. (1912). The Mechanistic Conception of Life. University of Chicago Press: Chicago. pp. 5-6.

[2] Pereto, J., Catala, J. (2007). The renaissance of synthetic biology. Biological Theory 2:128-130.

[3] Luisi, P. L. (2002). Toward the engineering of minimal living cells. The Anatomical Record 268:208-14.

[4] Szostak, J.W.; Bartel, D.P.; Luisi, P.L. (2001). Synthesizing life. Nature 409:387–390.

[5] Stano, P. (2019). Is research on “synthetic cells” moving to the next level? Life 9(1):3. doi:10.3390/life9010003

[6] Schrodinger, E. (1945). What is Life? Cambridge University Press: Cambridge, London, New York, New Rochelle, Melbourne, Sydney.

[7] Fleischbaker, G. R. (1990). Origin of life: An operational definiton. Origins of Life and Evolution of Biospheres 20:127-137.

[8] Rizzotti, M. et al., ed. (1996). Defining Life: The Central Problem in Theoretical Biology; University of Padova Press: Padova, Italy.

[9] Ruiz-Mirazo, K., J. Pereto, Moreno, A. (2004). A universal definition of life: autonomy and open-ended evolution. Origins of Life and Evolution of Biospheres 34:323-346.

[10] Bruylants G., Bartik, K., Reisse, J. (2010). Is it useful to have a clear-cut definition of life? On the use of fuzzy logic in prebiotic chemistry. Origins of Life and Evolution of Biosphere 40:137-143.

[11] Abel, D. L. (2010). Is life unique? Life 2:106-134.

[12] Stano, ibid.

[13] Luisi, ibid.

[14] Oparin, A. I. (1938). The Origin of Life. Translated by S. Morgulis. Macmillan: New York.

[15] Haldane, J. B. S. (1929). The Origin of Life. The Rationalist Annual for the Year 1929.

[16] Miller, S. L. (1953). A production of amino acids under possible primitive Earth conditions. Science 117:528-529.

[17] Pereto, J. (2005). Controversies on the origin of life. International Microbiology 8:23-31.

[18] Blain, J. C., Szostak. J. W., 2014. Progress toward synthetic cells. Annual Review of Biochemistry. 83:615-640.

[19] Luisi, ibid.

[20] Le Chatelier, H., Boudouard, O. (1898). Limits of flammability of gaseous mixtures. Bulletin de la Societe Chimique de France (Paris) 19:483-488.

[21] This procedure is the first step in the assay for β-galactosidase. See for example: Javor, G.T., Ryan A., Borek, E. (1969). Studies of the impaired inducibility in relaxed mutants of E. coli. Biochemical and Biophysical Acta 190:442 452.

[22] Jackson, R. W, J. A. DeMoss (1965). Effects of toluene on Escherichia coli. Journal of Bacteriology 90:1420-1425.

[23] Halegoura, S., A. Hirashima, J. Sekizauwa, Inouye, M. (1976). Protein synthesis in toluene treated Escherichia coli. European Journal of Biochemistry 69:163-167.