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Project Description

The Problem​

ESA, NASA and JAXA are not the only space programs, which plan to have manned space flights to the moon, mars and other destinations - several private companies like SpaceX and Blue Origin also have interest in humanities next step in space exploration. These space programs are gaining more and more interest and popularity amongst the world's population. However, space programs are extremely expensive and cost a lot of resources. Rocket launches emit a lot of black carbon particles into the stratosphere which leads to a temporal higher temperature in this area1. This in turn can cause the global overturning circulation to slow down1. But because of its major role in transporting carbon and heat between the oceans and the atmosphere, a change in this circulation can have an impact on our climate as well1.

For example, a heavy launcher, which uses a kerosene fueled engine, emits around ten tons of black carbon into the stratosphere during every launch1. Furthermore, it is very expensive to get one kilogram of payload into space. For example, it costs around $23.300 to get one kilogram of payload to the ISS2.

All these factors make a space mission really expensive and economically as well as ecologically demanding. Our Project wants to face these problems and provide a more flexible, cheaper, and sustainable solution to avoid unnecessary lift-offs to the space stations and therefore spending a huge amount of money. With our solution the astronauts can print their own equipment by using a 3D-printable bioink made by a certain bacteria and harden it by using an optogenetic inducement. The bioink, created by the bacteria, is made out of alginate and cellulose. This whole process could save a lot of resources and allows our astronauts to be more independent and flexible. Additionally, this project can be implemented in various ways in hard to supply regions on earth as well

Our Project Description​

Space explorations and the search for new habitable planets are currently gaining more popularity. Not only because technical progress allows us to go even deeper into space, but also because environmental changes, the growing population and, connected to that, dwindling resources force us to think about drastic actions. Seeing space as a possible future habitat seems to be unreal for now, but it could be an actual solution to many of our problems.

For that reason, the iGEM-Team Düsseldorf 2022 decided to focus on this new and exciting topic. After doing some research on this subject, we quickly noticed that space programs spend a huge amount of money because they have to send tons of tools and everyday supplies up to the space stations. Because most of the equipment only has a short lifetime or can only be used once, they have to constantly send more supplies to the stations. Not only does this make space flights financially unattractive, but it also hinders the space stations to be independent of earth.

For that reason, our project CosMIC – Microorganisms in Space - offers a key to have cheaper and more independent space flights in the future. With our solution, the astronauts only need to bring a few things with them. This includes a 3D-Bioprinter, which only has to be installed once, the bacteria Azotobacter vinelandii, media for the bacteria and a light source. These four things combined can significantly reduce the amount of equipment which has to be carried in the rockets. With the help of the bacteria, the astronauts can print a bioink in the form of any wanted tool and then harden it with blue light during the printing process. In order to achieve this, the bacteria A. vinelandii must produce alginate because this serves as a basis for the bioink. As soon as the bacteria produced enough alginate, the living bacteria with the alginate can be printed in any desired form. Afterwards, by using an optogenetic inducement and exposing the bacteria to blue light, these bacteria begin to produce cellulose which solidifies the material. To get a whole object, the process of printing one line and then hardening it by using blue light has to be repeated multiple times. Once all these steps are finished, the astronauts have their own printed equipment made of alginate and cellulose.

3D-Printing has a lot of benefits compared to conventional manufacturing. It is possible to create new tools in less time and within an only one-step production3. Moreover, 3D-printing can save a lot of resources by only using the necessary materials without generating a lot of waste products. To use a 3D-Printer, a fitting ink is needed. Alginate turns out to be a very popular polysaccharide often used as a bioink. “Due to its low cost, good biocompatibility, and rapid ionic gelation, the alginate hydrogel has become a good option of bioink source for 3D bioprinting”3. In addition to that, alginate is a biodegradable polymer and therefore can potentially be reused3. Usually, Alginate is being derived from algae3, but the speciesAzotobacter vinelandii can produce its own alginate as well4. For that reason, we chose A. vinelandii, a nitrogen-fixing and gram-negative soil bacterium5. Additionally, A. vinelandii is relatively safe to humans and, classified as a S1 organism. When A. vinelandii is producing enough alginate to form a biofilm, this biofilm can be used as a bioink for the printing process. Past research has shown that a specific gene called alg8, which codes for a catalytic subunit of the alginate polymerase, is an important control point to produce alginates with higher molecular weights6. A higher molecular weight usually causes the viscosity to increase, which can be very advantageous for creating a more stable bioink3. For that reason, we are planning to overexpress the alg8 gene in A. vinelandii to create a bioink with a greater viscosity. But before the bacteria embedded in the alginate can be printed, the bioprinter we are using for our project has to be improved. The Anet 8 3D-Printer, which we are using for our project, has to be modified into a bioprinter in order to print living bacteria. We used brackets for a spray self-printing and other materials to adjust it to our goal. Additionally, we optimized the code for the bioprinter to make it work. The bioprinter can then be used to print the living bacteria with the alginate, which as a whole serves as the bioink. Because the consistency of the bioink is more similar to gelatin than to plastic, another polymer has to be added to build a more solid material. As our second polymer, we chose cellulose because of its high elasticity as well as its relatively high mechanical strength7. Bacterial cellulose has already been used in medicine, dentistry, but also in the textile industry7. Another advantage would be that cellulose is a biodegradable polymer and therefore could be reused. In space, where water is limited and the astronauts do not have any possibilities to wash their clothes, reusable materials could be a solution and be integrated in a circle of producing and reusing cellulose or alginate7. Besides, paper and tissues are mostly made out of cellulose as well and could increase the sanitary standard of space related facilities. Because of all these reasons, we decided to use cellulose to make our material more stable and sustainable. Although these two polymers have not been used before together with a 3D-printer, we still decided to merge these two and create a new way of manufacturing.

But A. vinelandii does not produce cellulose naturally, for which reason we have to introduce the genes for cellulose synthesis in A. vinelandii. Essential genes for the cellulose production were identified and studied in Komagataeibacter xylinius8. The two subunits BcsA and BcsB are involved in the cellulose synthesis by forming the polysaccharide chain, the ß-D-glucan chain8. The other two subunits BcsC and BcsD are responsible for packing the ß-D-glucan molecules and then transporting them to the cell surface and organizing the cellulose fibers in a highly organized structure8. Besides these four subunits, there are also other additional enzymes involved in this process and are necessary for the optimal synthesis of cellulose fibers: β-glucosidase and endoglucanase are important to rearrange glucan fibers in the wrong arrangement. Other important components are the endo-beta-1,4-glucanase and cellulase-complementing proteins, which are essential for the correct structure of the cellulose fibers8.

To control the amount and time of cellulose production, we decided to use the pDawn optogenetic inducement, which can be activated by using blue light9. The pDawn plasmid contains a histidine Kinase YF1 and connected to that a light-oxygen-voltage, blue-light photosensor domain to perceive the blue light9. Without any light source, YF1 phosphorylates the FixJ, a response regulator. The phosphorylated FixJ activates the FixK2 promoter, which leads to a gen expression of the λ phage repressor cI. This results in the cI repressing the strong λ promoter pR throughout the multiple cloning site for which reason the genes cannot be read9. The YF1 and FixJ are constantly expressed from the LacIq promoter. But in the presence of blue light the YF1 does not phosphorylate the FixJ for which reason the repressor cI cannot inhibit the pR promoter. The active pR promoter enables the multiple cloning site to be expressed9. By inserting the cellulose genes in the multiple cloning site of the pDawn plasmid, we can control the activity of these genes9. As soon as the bacteria are exposed to blue light, they start to produce cellulose. This is important because the cellulose must be produced after the printing process of each layer to create a more solid material. After inserting the cellulose genes in this pDawn system, this plasmid can be implemented in A. vinelandii to produce cellulose on command by using blue light.

Future Applications​

As already mentioned in the beginning, our project does not only offer a promising solution in space, but also here on earth. For example, long-lasting scientific expeditions, aid organizations and other groups, which often have difficulties to be provided with the necessary resources, could benefit from our project. Most scientific expeditions like going to the arctic or areas which are difficult to supply have some similar problems like space missions. They have to carry a lot of load with them because the resources in these regions are usually very limited. Furthermore, it is very difficult to supply these scientific teams with essentiell resources. Therefore, printing their own equipment like pipette tips or tubes would be a huge advantage and make them more independent. Since implementing an optogenetically inducible system makes it possible to adjust the consistency of the printed equipment, it would also be possible to print hygiene products, such as toothbrushes, combs, menstrual cups or other products like earplugs. Additionally, aid organizations in countries with poor infrastructure could take advantage of our project. Especially in such organizations, being more independent and flexible would be very helpful. For instance, supplying aid organizations in war zones with important resources is always associated with risks. Consequently, printing their own equipment could minimize the supply trips to these aid organizations. In conclusion, our project could be a great solution to offer different groups here on earth as well as in space more independence and flexibility. Besides that, our project can be modified by implementing this system into cyanobacteria such as Synechococcus elongatus and therefore creating an even more sustainable and independent solution. Because S. elongatus is growing autotrophically by using sunlight and CO2, less resources are needed to grow these bacteria, therefore the use of these bacteria would offer an even more sustainable solution10.

  1. Maloney, C. M., et al. (2022). The climate and ozone impacts of black carbon emissions from global rocket launches. Journal of Geophysical Research: Atmospheres, 127(12). https://doi.org/10.1029/2021JD036373
  2. Jones, H. (2018). The Recent Large Reduction in Space Launch Cost. http://hdl.handle.net/2346/74082
  3. Gao, Q. et al. (2021). Advanced Strategies for 3D Bioprinting of Tissue and Organ Analogs Using Alginate Hydrogel Bioinks. Marine Drugs, 19(12), 708. https://doi.org/10.3390/md19120708
  4. Climenti, F. (1997). Alginate Production by Azotobacter Vinelandii. Journal Critical Reviews in Biotechnology, 17(4), 327-361. https://doi.org/10.3109/07388559709146618
  5. Castillo, T. et al. (2020). Respiration in Azotobacter vinelandii and its relationship with the synthesis of biopolymers. Electronic Journal of Biotechnology, 48(2020), 36-45. https://doi.org/10.1016/j.ejbt.2020.08.001
  6. Diaz-Barrera, A. et al. (2012). Alginate production and alg8 gene expression by Azotobacter vinelandii in continuous cultures. Journal of Industrial Microbiology and Biotechnology, 39 (4), 613-621. https://doi.org/10.1007/s10295-011-1055-z
  7. Kamiński, K. et al. (2020). Hydrogel bacterial cellulose: a path to improved materials for new eco-friendly textiles. Cellulose 27(2020), 5353–5365. https://doi.org/10.1007/s10570-020-03128-3
  8. Römling, U. et al. (2015). Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions. Trends in Microbiology 23(9), 545-557. https://doi.org/10.1016/j.tim.2015.05.005
  9. Ohlendorf, R. et al. (2012). From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. Journal of Molecular Biology (JMB) 416(4), 534-542. https://doi.org/10.1016/j.jmb.2012.01.001
  10. Yu, J., et al. (2015) Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO₂. Sci Rep 5, 8132. https://doi.org/10.1038/srep08132