Background

• The history of humanity is filled with exploration and courage. We live in a vast universe, but our current civilization is limited to Earth. This means our living space is finite, and we may face various unknown dangers in the future. Therefore, extending human civilization to other planets is crucial.
  
  Mars, one of the eight planets in the solar system, is about 400 million kilometers away from Earth at its farthest and about 55 million kilometers at its closest. Even at its closest, it is more than a hundred times the distance from Earth to the Moon. From the comedy “Uncle Martin from Mars” where a Martian accidentally falls to Earth, to the sci-fi movie “The Martian” where humans survive on Mars, this distant and mysterious red planet has sparked endless imagination. Since the 1960s, humans have begun exploring Mars. So far, more than forty Mars exploration missions have been carried out worldwide, achieving flybys, orbits, landings, and roving explorations of Mars.

Challenges on Mars

• Food:There is no natural food on Mars for humans to replenish energy, so all food must be brought from Earth. In the long run, we need to grow plant.
• Oxygen:The oxygen content on Mars is extremely low, with only 0.15% oxygen in the thin atmosphere, which is almost negligible. For the first batch of Mars colonists, plants that travel with them become a lifeline. These plants must be carefully tended in the specific environment of the Mars lander because they not only provide oxygen but also potentially food.
• Soil: Due to the presence of UV, the chlorides on the surface of Mars will constantly switch valence states, and most of them have antibacterial and plant growth inhibiting effects. This could have a significant impact on future Mars colonization plans.

Steps toward the solution

Cyanobacteria and Carbon Fixation

• Gradually reducing carbon dioxide and increasing oxygen content could potentially provide a breathable environment for future biospheres. The high carbondioxide content in the Martian atmosphere allows cyanobacteria to promote their growth while converting them into oxygen necessary for life. This process not only maintains the carbon cycle in the ecosystem but also provides organic matter and energy sources for other organisms. Additionally, cyanobacteria can adapt to harsh environments such as high salinity, strong light, and nutrient-poor conditions through special mechanisms.

Yeast and Space Food

• Yeast plays an important role in space food production, especially in providing sustainable nutrition for astronauts on long-term missions. Yeast is rich in protein, vitamins, and minerals, making it an important source of protein and B vitamins, helping astronauts maintain health. Through fermentation technology, yeast can improve the flavor of food and extend its shelf life, alleviating the psychological pressure caused by monotonous diets. Moreover, yeast can rapidly reproduce in space environments, requiring few resources, and is part of a closed-loop ecosystem that can recycle carbon dioxide and waste to produce food. Genetic engineering can also modify yeast to produce specific nutrients or medicines, further meeting astronauts’ needs. This makes yeast a key focus of future space food technology research.

Nitrogen-Fixing Bacteria and Plant Growth

• Nitrogen-fixing bacteria play a crucial role in plant growth, especially in providing the nitrogen plants need. Nitrogen is a key element for plant growth, involved in the synthesis of proteins, nucleic acids, and chlorophyll. However, atmospheric nitrogen (N₂) exists in a stable form that plants cannot directly use. Nitrogen-fixing bacteria can convert atmospheric nitrogen into ammonia (NH₃) that plants can absorb, promoting healthy plant growth.

De novo plant synthetic biology

• Genetic transformation stands as a pivotal realm within the domain of functional genomics research and molecular breeding within the realm of plant science. The focal point of genetic engineering, molecular biology, and genetic breeding revolves around the creation of secure, efficient, and innovative genetic transformation techniques. Conventional approaches for transforming plants encompass the use of Agrobacterium tumefaciens infection, particle bombardment, and viral infection. These techniques have been extensively applied in various model plants, such as Arabidopsis thaliana and tobacco, as well as annual or biennial herbs.

• In recent years, with the swift progress of nanoscience and nanotechnology during the latter part of the 20th century, nanomaterials have gained widespread adoption in nanobiology and gene therapy. Their appeal lies in their diminutive size, expansive surface area, biocompatibility, biodegradability, low toxicity, and reduced immunogenic properties.

Our design

• In the sixth year of our team participated in the iGEM competition, we decided to solve the problems in space exploration. Our team considered response from human practice throughout. Therefore, we come up with to four aspects to tackle martian immigrants after a series of human practice.
  
  Firstly, we decided to produce nutritious food by co-culture of engineered yeast and cyanobacteria. Secondly, to conserve the martian soil into farm land, we decided to isolate nitrogen fixing bacterica and demonstrate directed evolution to made it resist to toxin in Mars. Thirdly, we want to introduce a new toolkit which will speed up the DBTL cycle of plant synthetic biology to encourgae more iGEMer engage in plant synbio. Finally, we designed to use DNA storage to record representative things in the Earth.

Production of food additives using yeast and Cyanobacteria

• During our human practices, interviews with psychologists and nutritionists revealed that space explorers experience unique mental stresses not present on Earth. However, incorporating specific nutrients into their diets can help alleviate this stress.
  
  Traditionally, these substances are extracted from natural resources, but bioengineered yeast offers an ideal production system for space applications. We have chosen yeast CEN.PK2-1C strain to produce lycopene, a red food pigment with antioxidant and anti-UV properties. And also limonene and Nerol, two food essences also recommended by our experts in human practice.

  

  
  
  Carbon sources can be the major factor affecting the cost of fermentation production. Cyanobacteria can use carbon dioxide during photosynthesis, which occupies 95.3% in the air of Mars, and provides sucrose as carbon source for yeast.
  
  Our design includes expression of CscB to transport intracellular sucrose to extracellular environment, also improve this process by over expression of SPS and GlgC.

  

  
  
  What's more, as we mentioned in human practice, we decided to increase the growth rate of cyanobacteria by overexpression of KatG. This will encourage more iGEM teams to choose cyanobacteria as their chassis, and enrich the population of plant synthetic biology.

Evolutionized Nitrogen Fixation

• While producing food in a fermentor using engineered yeast is a potential solution, traditional soil-grown food better meets human mental needs. Therefore, we decided to genetically engineer the nitrogen-fixing bacterium Azotobacter vinelandii to degrade perchlorate, a major toxic component (0.4 to 0.6 wt%) to plants in Martian soil.

  

  
  However, none of the strains we purchased from commercial sources, including microbial depository centers, were correct. As discussed in our human practices session, Dr. Jian Huang suggested isolating chlorite-tolerant nitrogen-fixing bacteria from rhizobia inoculant and providing us with some samples from his lab. Meanwhile, we used E. coli to demonstrate that directed evolution, a plug-and-play method, can be used to improve bacteria's resistance to chlorite.

Multifunctional kill switch

• Prevent leakage of engineered microorganisms into the environment was important for biosafety. Besides regular approach of waste treatment, our two systems both contain a "suicide" part.
  
  For the food producing engineered yeast system, we designed vanillic acid-controlled suicide switch. Vanillic acid is a FDA-approved food addictive. Based on our design, expression of toxin is inhibited by TetR, and its expression is upstreamly inhibited by VanR. During the production process, vanillic acid will be added. It can release the inhibition of VanR to PvanCC, therefore expression of TetR will be strong, and it can inhibit the expression of toxin, and cell will not be kill. If engineer strain was released to the environment accidentally, without the presence of Vanillic acid, the VanR will express the expression of tetR, and expression of toxin will be strong, so strain will be kill.

  

  
  
  When applying engineered bacteria into the environment for treatment, a safety system is essential to confine the bacteria to their intended environment and prevent them from affecting the normal microbial community after treatment.
  
  Our new design includes using KillRed as a suicide switch, which releases reactive oxygen species to eliminate bacteria in a normal environment. We employ the Cre-loxP system to regulate KillRed's expression. Cre is a cyclization recombinase that interacts with loxP sites to reverse the DNA sequence between them when the sites are inverted. After incorporating a specific "sensor"—an inducible promoter triggered only in the intended environment, such as high salt concentration or low oxygen levels. This controls Cre, with the inducible promoter (Pinducible) determined by the environmental factors relevant to the application.
  
  So when we put the strain in an environment that needs to be improved, Cre will flip the promoter upstream of KillRed, so that Killred will not be expressed and the bacteria will not be killed by sunlight. At the same time, the promoter was flipped, and the expression of population control device (BBa_K3893030) was turned on to control the population number of bacteria.

  

  
  
  We believe that the design of these two suicide switches can serve as a reference for other iGEM teams, and they effectively address the risk of leakage of synthetic biology strains in both food and environmental topics.

Accelarated plant synbio

• Our team began working on agriculture-related projects since 2020. Despite our efforts, we're disappointed that few other iGEM teams are choosing topics in plant synthetic biology. Through our experience, we've become familiar with the common challenges in this field, such as the difficulty of performing experimental methods like tissue culture and Agrobacterium transformation. Additionally, growing model plants is both time-consuming and challenging, and the expensive equipment required for plant-related projects poses a significant barrier.
  
  Recognizing these obstacles, we decided to focus on providing solutions to help iGEM teams overcome these challenges and accelerate the Design-Build-Test-Learn (DBTL) cycle in plant synthetic biology. By addressing these issues, we aim to inspire more teams to explore and innovate in this vital area.

  

  

  
  Although conventional plant transformation methods like Agrobacterium-mediated transformation and the gene gun have been used for over 50 years in plant biology, and numerous protocols and commercial kits are available, these methods still face challenges. These include lengthy experiment times and limitations on the species that can be transformed.
  
  Beginning in 2023, our team introduced the Carbon Nanodot-based Tracked, Transformation, Translation, and Trans-regulation (TTTT) system and developed a project around it. By using carbon nanodots to deliver a gene circuit containing a low-phosphate-inducible promoter, a low-noise amplifier, and an optimized fluorescent protein into plants, we transformed the entire plant into a "phytosensor" capable of detecting phosphate starvation.

  

  

  
  This year, with the enhancement of carbon nanodot materials, our newly designed seed soaking method can achieve not only transient expression but also permanent expression through genomic insertion in plants. This indicates that the TTTT method can serve as a complementary option to traditional plant transformation techniques.

  

  
  Moreover, to accelerate the process and reduce the difficulty of cultivating model plants, we focused on the potential of using Wisconsin Fast Plants, a rapid-cycling Arabidopsis relatives developed by Professor Emeritus Paul H. Williams at the University of Wisconsin-Madison. These plants offer a much easier operation and a faster lifecycle of just 35 days compared to traditional model plants like tobacco and Arabidopsis. We hope this will lead to wider adoption in plant synthetic biology research.

References:

[1] Ranran Zhao, Y.M. John Chew, Jan A.M.H. Hofman, Holger V. Lutze, Jannis Wenk, UV-induced reactive species dynamics and product formation by chlorite,Water Research 2024
[2] https://www.reddit.com/r/ImagesOfThe1930s/comments/647cnz/cosmic_voyage_russian_space_art_1936/
[3] https://science.nasa.gov/resource/spirit-opportunity-20th-anniversary-poster/
[4] Llorente, B., Williams, T.C., Goold, H.D. et al. Harnessing bioengineered microbes as a versatile platform for space nutrition. Nat Commun 13, 6177 (2022). https://doi.org/10.1038/s41467-022-33974-7
[5] Maity, Tanushree, and Alok Saxena. "Challenges and innovations in food and water availability for a sustainable Mars colonization." Life Sciences in Space Research (2024).
[6] Xie, Xiulan, et al. "Microalgae: towards human health from urban areas to space missions." Frontiers in Plant Science 15 (2024): 1419157.
[7] Ute Krämer (2015) The Natural History of Model Organisms: Planting molecular functions in an ecological context with Arabidopsis thaliana eLife
[8] https://2020.igem.org/Team:SZ-SHD
[9] https://2023.igem.wiki/sz-shd/engineering
[10] Oze, Christopher, et al. "Perchlorate and agriculture on Mars." Soil Systems 5.3 (2021): 37
[11] Carrier, Brandi L. "Next steps forward in understanding Martian surface and subsurface chemistry." Journal of Geophysical Research: Planets 122.9 (2017): 1951-1953.