Oil droplets Synthetic biology Why endowing organisms with unnatural functions demands a better understanding of biology, designer proteins, and some soul searching.. Oil droplets in water, a common approach for creating lipid-enclosed ‘protocells’. Photograph by A_Different_Perspective Pixabay. See: https://www.nature.com/articles/ncomms6305

Synthetic Life: Made from Scratch

Synthetic biology is a scientific discipline that aims to rationally engineer living organisms, typically with genetic engineering approaches (1). In 1961, Francois Jacob and Jacques Monod first proposed that genetic regulatory circuits direct cellular behavior (2). By 2000, scientists successfully engineered unnatural genetic circuits, implanted them into microorganisms, and the circuits carried out their specified function. Early examples include the genetic toggle switch (3), wherein two promoters drive the expression of mutually inhibiting repressors, causing the cell to ‘toggle’ between steady states, and the repressilator (4), which emerged in the same year.

Genetic circuits Synthetic biology
Genetic circuits are inspired by electrical circuits and are built from similar principles, by geralt Pixabay.

Today, the application of engineering methodologies to the rational modification of organisms is a persistent goal of synthetic biology. Most synthetic biologists describe biological engineering as a hierarchy, wherein parts (genes, DNA) are used to build devices (many genes together), which in turn can be used to construct systems (a series of many devices) (1). The challenge in transforming synthetic biology into a true engineering discipline is that the parts, which are the rudimentary building blocks of higher-order constructions, are fundamentally limited by the rigor of their characterization. This is really the case in all established engineering disciplines. In electrical engineering, for instance, the baseline components (transistors, resistors, wires, etc.) have been characterized so well that children can use them and the resulting circuits behave as expected. Once all ‘parts’ are standardized, it may be possible for synthetic biologists to use individual DNA building blocks to construct entirely synthetic life forms from the bottom-up.

Aristotle
Bust of Aristotle. Photograph by morhamedufmg Pixabay.

The idea of synthetic life has existed for millennia. Aristotle, in the 4th century BC, wrote about spontaneous generation in his book, ‘On the Generation of Animals’, which purported that decaying flesh yielded entirely new life forms. By the 20th and 21st centuries, serious ethical concerns surrounding artificial life forms arose. In 2005, the bacteriophage T7 virus was successfully ‘refactored’ by replacing 11,515 base pairs of DNA with a synthetic form and the viability of the viral particles maintained (5). Two years later, J. Craig Venter succeeded in transplanting chromosomes between microorganisms (6) and, the next year, published a completed artificial genome based on M. genitalium (7). In 2010, the components were pieced together and an M. genitalium containing a ‘synthetic’ genome was successfully constructed (8).

An ongoing, international project called Synthetic Yeast 2.0 is attempting to construct the first eukaryotic organism possessing a chemically-synthesized genome (9). The most ambitious example of synthetic genome construction to date, each member institute is constructing and troubleshooting one of the 16 yeast chromosomes. In the next few years, they hope to produce a fully ‘synthetic’ yeast possessing all of these chemically-synthesized chromosomes.

Last year, the Romesberg group at The Scripps Research Institute published the creation of a ‘semi-synthetic life form’, which provided the first evidence of a microorganism with synthetic nucleotides in its genome (called X and Y), the code of which was successfully transcribed and translated, thus expanding the amino acids available to living cells from 20 to 172 (10). Romesberg was careful in conveying his results to the media, stating, “I would not call this a new life form — but it’s the closest thing anyone has ever made” (11).

The closest effort today to constructing something that can truly be called synthetic life is the Build-a-Cell consortium, which aims to build synthetic cells from the ground-up using modular components. Theoretically, a cell possessing all the genes necessary for basic metabolism, cell division, signaling and a few other tasks could be considered alive, and constructed entirely from well-characterized building blocks.

But what about the development of organisms that possess entirely new functionalities — those not found anywhere in nature? How can synthetic biologists transition beyond the rewiring of existing components (which has only partially been achieved) and move into the realm of unknowns? There are many challenges limiting this transition, but it will happen eventually. An artificial life form, one that is only loosely based on an existing organism, can only be constructed if its developer has a complete understanding of how life operates and can predict how each component will behave within the cell. Since proteins are one of the most important means by which a cell exacts its functions, it stands to reason that an improved understanding of protein function, and the ability to design proteins with entirely new functions, could facilitate this transition.

There are three main ‘challenges’ in developing synthetic life forms with new functionalities. Though the examples provided here are in no way exhaustive, they encompass the scientific, technological and ethical.


Scientific Challenge: Unknown Protein Functions

Synthetic biology is a discipline reliant on parallel advancements in genomics, molecular biology, and computation. To engineer organisms in predictable ways, a firm grasp of their intricacies, differences and, importantly, the function of each component, must be understood before components can be rearranged and transplanted at will. This is the scientific challenge.

To find a compelling example of the existing, glaring gaps in scientific knowledge that need to be addressed before creating life anew, one need look no further than the efforts to build a minimal genome, completed in 2016, in which the genome of Mycoplasma mycoides was reduced to a mere 473 genes (12). It is perhaps surprising that this effort was preceded, in 2012, by the creation of a whole-cell model, based on the simple organism Mycoplasma genitalium, by Markus Covert’s group at Stanford (13). Despite the simplicity of these organisms (Native genitalium only has 525 genes) and the intense scientific efforts devoted to understanding them, 149 essential genes in M. mycoides have an unknown function (12).

A fundamental limitation in constructing synthetic life forms is that the main method of conducting biological research, historically, has been to isolate or knock-out genes independently and then identify their function within the cell. This approach is useful, but may be flawed to investigate all of the complex functions it is involved in, as “…a single component (such as a gene) rarely specifically controls any particular biological function or disease, and conversely any given component may influence many different functions” (1). To understand how components within the cellular milieu perform their functions, canonical biochemical approaches are time-consuming and laborious, but they may remain as a necessity. Other systems-level approaches have been used to predict function from sequence, however.

In the months that followed Venter’s publication of a viable, minimal cell, Antoine Danchin and Gang Fang predicted some of the unknown gene functions via evolutionary analysis, scouring through the literature to determine essential genes that perform basic functions in related bacterial clades which were not mentioned as one of the known genes in the minimal genome mycoides(14). Using these evolutionary relationships, Danchin and Fang proposed identities for 32 of the 149 unknown genes (14). But the minimal cell (and its predecessor) are not the only relatively simple organisms that have been extensively studied, and evolutionary relationships are not the only way to study protein function. Yeast researchers have long sought new methods to determine the function of unknown genes. In 2007, there were over 1000 uncharacterized genes in yeast (15).

It is one thing to perform homology or evolutionary analyses on large datasets, but quite another to isolate the protein in question and perform enzymatic studies or delete the gene in vivo to study its effects. With essential genes, the deletion is harder to obtain results from, but there are still options available to study protein functions, such as fluorophore tracking, immunoprecipitations, immunoblotting and pharmacological inhibitors. Though time consuming and laborious, perhaps a biochemical approach based on proven tools is still the best method to determine function.

Once the function of each component is determined, the next step is to use systems-level approaches to understand how they function within the greater cellular context. This is the job of systems biologists, who “seek to understand how all the individual components of a biological system interact in time and space to determine the functioning of the system. It allows insight into the large amount of data from molecular biology and genomic research, integrated with an understanding of physiology, to model the complex function of cells, organs and whole organisms” (16). Today, a limited understanding of protein function is severely hindering the construction of synthetic life.


Technological Challenge: Predicting Function from Sequence

In the future, it would be useful to create cells that carry out a tailor-made purpose, even if that purpose demands an enzymatic function or behavior not found anywhere in nature. With advancements in the prediction of protein structure and function from DNA sequence, the ‘modularity’ of parts that could one day be used to construct biological organisms from the bottom-up will be drastically expanded. While some research groups are working towards the creation of proteins with completely novel functions, including 2018 Nobel Laureate Frances Arnold’s group, a better understanding of protein function and tools to reliably engineer protein structure are desperately needed. A major technological challenge related to the creation of entirely ‘unnatural’ organisms is: what methods can be used to build proteins with novel functionalities and in what ways are we constrained?

The ‘protein folding problem’ has been tackled by many groups, perhaps none as famous as David Baker at the University of Washington. Baker’s group is notable (partly) for their development of ROSETTA and ROSETTA@Home, a protein structure prediction program, and its version for home use, in which people at their personal computers can work to solve the lowest energy structure for a variety of proteins (17). Baker has also created a spin-out company, called Arzeda, which uses structural prediction platforms for the creation of enzymes with new functionalities that can be used for environmental, diagnostic and therapeutic applications (20).

Computational programs developed by the Baker group and others have been used to design a completely unique protein fold not found in nature (18) and to design protein-protein interfaces for applications in therapeutic design (19). But one current limitation in the design of entirely novel biological structures is computing power. Ab initio simulations typically probe and test many conformational protein states to identify structures with the lowest free energy (20, 21). In 2009, a state of the art supercomputer could simulate a 50-residue protein, atom-by-atom, for 1 millisecond. This capability has since been exceeded by personal computers (22, 23).

Unfortunately, the prediction of protein function from a DNA sequence is far more complex than predicting protein structure. The ability to predict protein functions could enable the rapid, rational design of proteins with entirely new activities. Frances Arnold’s group at the California Institute of Technology tackles this problem by using the “most powerful biological design process, evolution, to optimize existing enzymes and invent new ones, thereby circumventing our profound ignorance of how sequence encodes function.” Evolution is such a great tool to develop new proteins, in part, because the mutations necessary to implement a useful new function are often highly non-intuitive. Though most amino acids responsible for substrate specificity or selectivity are found in the active site, alterations to amino acids distant from the active site can also result in drastically enhanced catalytic activities (24).

A culmination of approaches encompassing computation, design, and evolution are most likely to succeed in creating ever more complex proteins by design.


Societal and Ethical Challenge

The ethics of synthetic biology has been hotly contested since the first reports of genetic circuits published in the early 2000s, and the report of a chemically-synthesized genome in 2010 prompted the Obama administration to create a bioethics commission to address new capabilities in synthetic biology (25). During this initiative, ethicists claimed that, if scientists were to succeed in creating the organism, life itself could lose its special status (26). In other words, people would begin to view life as nothing more than a series of intricate biochemical reactions that can be replicated in a laboratory, and the creation of a reductionist, synthetic organism would undermine this special status.

The reductionist moral argument against the creation of synthetic life is also related to methodological reductionism, a strategy which engineering disciplines have exploited to systematically reduce components to further understand how the whole is built. This is the case for standardization in synthetic biology, in which each component is individually characterized and, yet, there has been no backlash ethically over systematic biological standardization. These are, however, the same reductionist principles that will enable the eventual creation of a synthetic life form. Finally, there is no evidence to suggest that previous efforts to build semi-synthetic life, mentioned earlier in this article, and organisms with synthetic genomes, has in any way damaged the special status that humans ascribe living organisms despite the media attention.

Nevertheless, there are key ethical points to consider before synthetic life forms are built. The guidelines put forth by Weitze and Pühler are particularly insightful (27). First, do scientists have all relevant knowledge and a comprehensive understanding of the technology in question? Just because scientists can build something does not mean that they should. Accordingly, scientists should work towards a full understanding (where possible!) of the biological principles at play within the organism so that potential problems can be prevented or mitigated.

The potential detriments of a novel synthetic organism must also be considered. People often use technology in unexpected ways, so it is important that potential applications of the synthetic organism be debated proactively. Government regulations and ethical considerations should be accounted for long before actual construction is initiated.

The framework of responsible research and innovation, developed by the EPSRC (Engineering and Physical Sciences Research Council, the UK’s main funding agency for engineering and the physical sciences), calls for synthetic biology research to be conducted using AREA, which stands for Anticipate, Reflect, Engage and Act (28). Researchers should fully explore the impact of their research project before embarking on it, reflect on the purposes for carrying out the research, engage with people outside of their own discipline, including bioethicists, and then act on these processes and shape the direction of their research project accordingly.

In the decades to come, cells with entirely unique, unnatural functionalities will be designed and constructed. Advancements made today are stepping stones towards this greater aim, and may usher in a new era of synthetic biology, where organisms are made ad hoc to address some of the world’s most pressing problems.


References

1. “Synthetic Biology: scope, applications and implications.” The Royal Academy of Engineering. May 2009. Accessed 2 December 2017.

2. Jacob F and Monod J. “Genetic regulatory mechanisms in the synthesis of proteins.” Journal of Molecular Biology 3, 318–356 (1961).

3. Gardner TS, Cantor CR and Collins JJ. “Construction of a genetic toggle switch in Escherichia coli.” Nature 403, 339–342 (2000).

4. Elowitz MB and Leibler S. “A synthetic oscillatory network of transcriptional regulators.” Nature 403, 335–8 (2000).

5. Chan LY, Kosuri S and Endy D. “Refactoring bacteriophage T7.” Mol Syst Biol 1 (2005).

6. Lartigue C, Glass JI, Alperovich N, et al. “Genome transplantation in bacteria: changing one species to another.” Science 317, 632–8 (2007).

7. Gibson DG, Benders GA, Andrews-Pfannkoch C, et al. “Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome.” Science 319, 1215–20 (2008).

8. Gibson DG, Glass JI, Lartigue C, et al. “Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome.” Science 329, 52–6 (2010).

9. Annaluru N, Muller H, Mitchel LA, et al. “Total Synthesis of a Functional Designer Eukaryotic Chromosome.” Science 344, 55–8 (2014).

10. Zhang Y, Ptacin JL, Fischer EC, et al. “A semi-synthetic organism that stores and retrieves increased genetic information.” Nature 551, 644–47 (2017).

11. “Scientists Create First Semi-Synthetic Organism that Stores and Retrieves Unnatural Information.” The Scripps Research Institute News Release. 29 November 2017. Accessed 2 December 2017.

12. Hutchison CA, Chuang R, Noskov VN, et al. “Design and synthesis of a minimal bacterial genome.” Science 351, 1414 (2016).

13. Karr JR, Sanghvi JC, Macklin DN, et al. “A Whole-Cell Computational Model Predicts Phenotype from Genotype.” Cell 150, 389–401 (2012).

14. Danchin A and Fang G. “Unknown unknowns: essential genes in quest for function.” Microb Biotechnol 9, 530–40 (2016).

15. Peña-Castillo L and Hughes TR. “Why Are There Still Over 1000 Uncharacterized Yeast Genes?” Genetics 176, 7–14 (2007).

16. “Systems Biology: A Vision for Engineering and Medicine.” Academy of Medical Sciences and Royal Academy of Engineering (2007).

17. Kaufmann KW, Lemmon GH, DeLuca SL, Sheehan JH and Meiler J. “Practically Useful: What the ROSETTA Protein Modeling Suite Can Do for You.” Biochemistry 49, 2987–98 (2010).

18. Kuhlman B, Dantas G, Ireton GC, Varani G, Stoddard BL and Baker D. “Design of a novel globular protein fold with atomic-level accuracy.” Science 302, 1364–68 (2003).

19. Ashworth J, Havranek JJ, Duarte CM, et al. “Computational redesign of endonuclease DNA binding and cleavage specificity”. Nature 441, 656–59 (2006).

20. Jiang L, Althoff EA, Clemente FR, et al. “De novo computational design of retro-aldol enzymes.” Science 319, 1387–91 (2008).

21. Simons KT, Bonneau R, Ruczinski I and Baker D. “Ab initio protein structure prediction of CASP III targets using Rosetta.” Proteins 37, 171–76 (1999).

22. Shaw DE, Bowers KJ, Edmond C, et al. “Proceedings of the Conference on High Performance Computing Networking, Storage and Analysis — SC ‘09.” (2009).

23. Pierce LCT, Salomon-Ferrer R, de Oliveira CAF, et al. “Routine Access to Millisecond Time Scale Events with Accelerated Molecular Dynamics.” J Chem Theory Comput 8, 2997–3002 (2012).

24. Tracewell CA and Arnold FH. “Directed enzyme evolution: climbing fitness peaks one amino acid at a time.” Curr Opin Chem Biol 13, 3–9 (2009).

25. Pollack A. “U.S. Bioethics Commission Gives Green Light to Synthetic Biology.” The New York Times. 16 December 2010.

26. Cho MK, Magnus D, Caplan AL, and McGee D. “Ethical considerations in synthesizing a minimal genome.” Science 286, 2087–89 (1999).

27. Weitze M and Pühler A. “Synthetic Biology — Towards an Engineering Science.” European Review 22, S102–12 (2014).

28. “Framework for Responsible Innovation.” Engineering and Physical Sciences Research Council.

Originally published on Medium https://medium.com/@NikoMcCarty/synthetic-life-forms-could-change-everything-so-why-hasnt-it-been-done-e79c398ea657

Niko McCarty

Niko McCarty

Science | Tech | Bioengineering PhD Student @ CalTech | Formerly SynBio at Imperial College London

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