Synthetic biology meets bioprinting: enabling technologies for humans on Mars (and Earth)

Imagine a technology that has the following properties. It is programmable like a computer, and modular in design. The technology is self-replicating so multiple units are available for the cost of one. Damage is not an issue as it is self-repairing. It can perform advanced chemical transformations in a tiny form factor and in a non-toxic manner at room temperature and near neutral pH. Its abilities in the field of nanotechnology are unparalleled, with atomic scale precision assembly a nearly constant activity. It can sense as little as a single molecule. Its energy requirements are modest, and never involve petrochemical or electrical sources. In fact, some have built in solar converters whereas others use inorganic energy sources such as hydrogen sulfide (H₂S), elemental sulfur, ferrous iron (iron II), molecular hydrogen (H₂), manganese (Mn²⁺) and ammonia (NH₃). However, some can go for periods of time in excess of a century without any energy input. There are probably over 9 million variants available today [1,2].

This technology is, of course, life.

Once we begin to think of biology as a technology, the potential applications are stunning. For example, the materials that are produced by life have an array of mechanical properties unrivalled by either natural or man-made products. For example, spider silk is reputed to have a tensile strength greater than steel. Although it does fall in the range of steel (0.2–2 GPa), its stiffness is less than steel but its density is almost six times less. As a result, the strength to density ration of spider silk exceeds that of steel [http://phys.org/news/2013-06-spider-silk-nature-stronger-steel.html#jCp]. And then think of other structural marvels of nature, from wood to bone, from fibres to shells.

We are now in an era where we can engineer microbes to make materials that were previously the exclusive domain of larger creatures. This allows us to control the content of the materials through synthetic biology, and position the deposition of the materials through additive manufacturing and cell activation. Bone is wonderful, but production is limited by evolution and ethical sensibilities on the Earth and absent on Mars. But what if bone could be made without animals and laced with spider silk? Spider silk itself has been laced with carbon nanotubes and graphene flakes to produce an enriched spider silk that has a tensile strength greater than that of synthetic fibres such as Kevlar, making it the strongest fibre known [4].

Although such ‘not-as-futuristic-as-one-might-think’ ideas would no doubt benefit terrestrial industries, they may well be the key to enabling long-term human space exploration and colonization.

How will humans on Mars view planet Earth? Will that tiny dot in the sky (Figure 1) be seen as their real home, an ancestral home, a honeymoon destination or something less emotionally laden, like a remote data processing centre? What we do know is that the community will be physically independent.

The requirements of a human settlement will need to be met on location. The colonists will need transportation, not only to and from Earth, but on Mars itself for moving themselves, goods and reconnaissance. As with any other settlers, they will need habitats, clothing, food, water, medicines, waste removal and recycling. Although the solar output will be a resource, they will need supplemental sources of power, heat and light. Unlike the Earth, atmospheric oxygen is, for all practical purposes, absent at 0.15% molar fraction. Further, the atmospheric pressure averages 7.5 mbar in contrast with 1.013 bar on the Earth. Even though Mars is one and a half times as far from the sun as the Earth, the absence of an ozone ‘shield’ means that radiation protection is necessary. And, of course, gravity is far lower, approximately 38% that of Earth. Perchlorate (ClO₄⁻) is widespread on the surface, which is a potential source of oxygen as well as a conceivable health hazard [5].

There are challenges to supplying these needs, starting with upmass. Anything launched into space is expensive as it has to overcome Earth's gravity. Today it costs approximately $10000 to put a pound (454 g) of payload into Earth orbit. To visualize what this means, this is the same weight as a 16 oz can of soda, and it is approximately the official weight of a European (FIFA's Law of the Game number 2) or American (NFL Official Playing Rules of the National Football League 2015, rule 2) football. Similarly, ‘upvolume’ is limited as it too can affect upmass, so volume should be reduced to balance payload needs. Both upmass and upvolume are thus tied closely to the cost of the mission. Further, the technologies used must to be stored until needed, flexible in their applications and reliable as resupply will be infrequent.

Biology, and synthetic biology in particular, can overcome many of these challenges. The notion is that upmass and volume will be alleviated, as the organisms ‘live off the land’ through in situ resource utilization (ISRU). Although biology has produced a wealth of potential resources, none has evolved for life on Mars or for the needs of human Mars missions. Rather than wait for evolution, synthetic biology allows us to circumvent evolutionary time scales by producing bespoke organisms.

So what is the ‘big idea’? For millennia we have used biology to do chemistry on Earth. In the future, we will use biology to do chemistry beyond Earth, including material synthesis and recycling. We may use synthetically-altered organisms for material production including habitat construction, food, fuel, clothes and drug production, embedding biosensors. We may use materials acquired from Earth or acquired in transit (e.g. from an asteroid or repurposed upmass from missions) or recovered in situ through biomining. For exploration we will rely on nanotechnology, and what better way than to exploit the best nanotechnology production platform, living organisms. As we do in the lab today, we will send the information to synthesize new DNA constructs digitally overcoming the time delay of physical transport so that the DNA can be synthesized on site, allowing the just-in-time production of designer drugs. Life on Earth has chirality with left-handed amino acids and right-handed sugars. Why not produce mirror image cells, a ‘Life 2.0’, that uses an alternate biochemistry such as right-handed amino acids and left-handed sugars, thus preventing cross-contamination with terrestrial life? Why not use chemical energy to generate and store electricity as does the electric eel, or indeed all organisms with nervous systems?

Our lab at NASA Ames Research Center, located in Silicon Valley, has focused on creating proof-of-concepts as we prepare the way for future human voyages. Starting with 2011, some of these projects have been initiated by the Stanford-Brown iGEM team, a team of undergraduates who design, conduct and present projects for the annual international Genetically Engineered Machine competition (iGEM.org). With the goal of space exploration, we have the incentive to focus on reducing mass, autonomy and systems that operate in extreme environments. Decoupling from the economic and geopolitical realities of current Earth-based economies allows us to pioneer technologies that will revolutionize life for the humans who remain on Earth.

Biological materials are materials produced or derived from living organisms. This includes an amazing diversity of resources including biomolecules, biopolymers and hard structures that result from the deposition of minerals by biomineralization. Although some biological materials are useful in solution, such as oils, our focus here is on structural materials which may be created by bulk materials but often by materials arranged in a hierarchical structure.

Biologically-created structural materials have been critical throughout human history, from wood for construction, furniture and heat, to bone for knives and needles, fibres and leather for clothes and so on (Figure 2, left). There is no reason not to continue to use them beyond our home planet. At this point no one is suggesting transporting herds of sheep or setting up sugar cane plantations off planet. However, there is no reason not to take these biological capabilities with us but in a more tractable form factor (Figure 2, centre/right). Thus, rather than harvesting latex for rubber from a forest of 30 m tall Hevea brasiliensis trees, why not engineer a yeast cell to produce the rubber hydrocarbons from carbohydrates directly? This also may have the benefit of reducing antigenicity by not producing the additional compounds that are found in natural latex, including proteins, resins, sugars, glycosides, tannins, alkaloids mineral salts and secondary metabolites. Two leading tire manufacturers, Goodyear and Michelin, have entered into partnerships with a handful of biotech companies including Genencor to supply microbially-produced five carbon isoprene to make a synthetic latex similar to rubber [6,7]. Whereas on Earth the microbes will have to prove they are more economical than tree and petrochemical-derived rubbers, on Mars they will be the only option.

Biocomposites are defined as composite materials formed by the reinforcement of a matrix by natural fibres. But in a general sense, they could be considered materials that combine biological and non-biological components. Examples include the agglutinated tests (shells) of some amoebae (e.g. members of the Foraminifera and the rhizopod Heleopera petricola) which are composed of foreign particles glued together with a calcareous or organic cement. The organo-sedimentary laminated structures that make up stromatolites also could be considered biocomposites.

Harnessing the ability of organisms to create concrete has suggested a way to make building materials based primarily on minerals found on the surface of Mars, the moon or Earth. The bacterium Sporosarcina pasteurii (previously Bacillus pasteurii), can raise the pH of a solution of urine, sand and calcium chloride, thus inducing calcite precipitation. The microbially-induced calcite precipitation (MICP) has been used to create bricks. At least one company, bioMASON, produces bricks commercially by this procedure. The 2011 Brown-Stanford iGEM team [http://2011.igem.org/Team:Brown-Stanford/REGObricks/Biocementation], under our direction, pioneered the concept of using this approach to agglutinate the Martian regolith (surface material) to produce a brick. With that buildings could be built to provide wind and radiation protection. Even if the buildings then require additional infrastructure such as a vapour barrier, much of the mass of the structures would have been created using in situ materials.

Biological materials may be present in bulk or in aqueous suspensions such as blood and urine, but many biological materials derive their structural features by the organization of the components in a hierarchical structure based on a few polymers. For example, keratin is a protein in a family of fibrous structural proteins that are a major component of such diverse structures as skin, hair, nails, hooves, horns and teeth. They are intermediate filaments (average diameter of 10 nm) that derive properties from their hierarchical structure [8].

Seven features distinguish biological materials from their synthetic counterparts [9]. These are:

1. Self-assembly.

2. Multifunctionality where many components serve more than one function in the organism such as feathers serving for warmth, camouflage, sexual display and flight.

3. Hierarchical structure where the nano- to ultra-scale creates unique properties because of their relationship.

4. Hydration, a property nearly absent from materials such as ceramics, but the general rule for biology where water is the solvent for life. As a practical consequence, mechanical properties such as strength are decreased by hydration, whereas toughness is increased.

5. Mild synthesis conditions compatible with life, which for most organisms mean at temperatures between 10 and 40°C, and pH near neutral.

6. Evolutionary and environmental constraints which means that although some structures may have been optimized as the result of selection, evolution does not test all options nor is the optimal functional design of a feature necessarily selectively advantageous to the organism as a whole. To re-iterate the Jacob's point: evolution is a tinkerer, not an engineer [10].

7. Self-healing, a property critical to the Darwinian success of organisms.

Nearly of these properties can be retained, or some cases enhanced, by moving production into a microbial form factor if it does not already exist. The one exception is that microbial production cells in bulk do not produce large scale hierarchical structures. How can a microbe produce a feather, which demands macroscale assembly? Or the hierarchical structure of a toucon's beak [11] or of ivory [12]? One solution our laboratory has developed is to print the production cells in a pre-determined array, and then induce the cells to produce the structural material, as described in [13]. In theory, cells could be printed that then produced a sheet of bird's beak, a fabric with embedded biosensors or even a complete tool. Once this is realized, novel ‘synthetic biomaterials’ could be produced that will have no analogue in the living world, at any scale, anywhere. Imagine a yeast cell depositing a metallic filament, surrounded by a 5–10 μm layer of microbial cellulose, surrounded by cross-linked keratin, surrounded by silicate–a structure never found in nature but in theory could be produced with such a system.

The microbes that will serve as the production organisms for biological materials off planet will need inputs in order to power their metabolism. The basic nutritional types are primary producers (autotrophs), herbivores who eat the autotrophs, and carnivores who in turn eat the herbivores. The decomposers degrade biological materials produced by the first three. All four nutritional types are found among the microbes, and all four produce materials of use (Figure 3).

On Mars the primary inputs available are atmospheric gasses, primarily CO₂ (95%) with some N₂ (<2%), water mostly in the form of ice, surface minerals and sunlight. But, for the most part, the microbial ‘chassis’ organisms used in synthetic biology are decomposers and thus require an external source of organic carbon. We suggest that, as on Earth, a photosynthetic organism will be the interface between the raw materials and a food source for the production organisms. Diazotrophic (nitrogen fixing) cyanobacteria are ideal as they can covert N₂ to NH₃ as well as convert CO₂ to organic material. In 2011 the Brown-Stanford iGEM team suggested that this cyanobacterium would function as the metabolic power centre for the biological infrastructure, thus providing the inspiration for its name, PowerCell (http://2011.igem.org/Team:Brown-Stanford/PowerCell/Introduction).

The overall vision for a biology-enabled Mars colony consists of several components (Figure 4), alone or in combination. Solar radiation supplies the energy for PowerCell to convert in situ resources (CO₂, N₂, water and minerals) into organic compounds such as sugars and proteins. The products of PowerCell in the form of excreted organics or a cell lysate are then used as the feedstock for production organisms. These are microbes that have evolved to produce products of use such as the production of microbial cellulose by the bacterium Gluconacetobacter xylinus, previously known as Acetobacter xylinum and since reclassified as Komagataeibacter xylinus. Alternatively, the production organisms may have been genetically engineered for transgenic production, as in the microbial production of spider silk. If a hierarchical structure is desired, the production cells could be printed in specified arrays and then production and secretion of the product initiated. The resulting products could range from clothing to aircraft parts, construction tools to medical devices.

It should be noted that although the focus of this article has been on production by microbes, surely small plants could be pressed into service. There has even been a suggestion of bringing small animals such as silkworm on crewed missions to Mars, both for material production and for food [15].

Currently the transit time from Earth to Mars is approximately 7 months. In addition to all the other constraints, organisms making the journey will be subjected to Earth gravity (1 × g), microgravity en route and approximately 0.38 × g at destination. How will this affect the vision of a synthetic biology-enabled settlement? Will we have to compensate for gravity when we conduct genetic engineering beyond Earth? How will gravity affect the efficacy of PowerCell? Production organisms? Bioprinting?

To address all but the last question, we will fly the PowerCell payload on the DLR (German Space Center) EuCROPIS satellite, with a scheduled launched of March 2017. During the mission the satellite has periods of time when the rotation rate will mimic microgravity, lunar gravity and finally, Martian gravity. Our NASA secondary payload will consist of two types of experiments, both conducted in microfluidic cards remotely. The PowerCell experiment will test the ability of the diazotrophic cyanobacterium Anabaena to feed a production bacterium, Bacillus subtilis, which will act as a reporter system as originally conceived by the 2013 Stanford-Brown iGEM team (http://2013.igem.org/Team:Stanford-Brown/Projects/EuCROPIS). The second set of experiments will consist of a basic transformation of the B. subtilis in space. As the samples will need to be loaded, stored dry with a trip from California to Germany and back prior to launch, systems have been developed for long-term stasis. With the engineering constraints on the payload, transformation protocols have been developed that obviate the need for electroporation or temperature changes.

Thus, by the end of the decade, we will have taken the first steps towards realizing the vision of a synthetic biology-enabled future off planet.

I wish to acknowledge the postdocs, graduate students and iGEM teams that have worked in my lab to pioneer the work presented. Drs Kosuke Fujishima and Ivan Paulino-Lima have been critical to the success of nearly all the projects mentioned. Dr Diana Gentry developed the bioprinting project as part of her Ph.D. thesis with the help of Kosuke and Ashley Micks, and Jesica Navarette is developing the BioMining project for her Ph.D. thesis. The 2011 Brown-Stanford team were the first to tackle the idea of creating a synthetic biology-enabled human Mars colony. I am particularly indebted to the contributions of Ryan Kent, Griffin McCutcheon, Evie Pless, Ryan Gallagher and Eli Moss for work on PowerCell since its inception as part of the 2011 Brown-Stanford iGEM team, the design of the initial bioreporter by the 2013 Stanford-Brown and the continuing work on the PowerCell payload for the DLR EuCROPIS satellite mission. I thank S. Pete Worden for his initiative in developing a programme in synthetic biology for NASA whereas Center Director of Ames and his financial support of the projects described here, and Dr John Cumbers for his vision of synthetic biology on Mars while a graduate student in my lab. And lastly, I thank Dr Vitor Pinhiero for his kind invitation to present this work at Synthetic Biology UK 2015.

This work was supported by the NASA Ames Research Center Director's Discretionary fund; NASA Headquarters; Brown University; the Stanford University; the Rhode Island Space [NASA Grant NNX10A195H, NASA Grant NNX15AI06H]; the Center Innovation Fund; the NASA Innovative Advanced Concepts programs; and the NASA's Advanced Exploration Systems program.