Description

Summary

Microorganisms face unique challenges to growth in space, requiring both protection from radiation damage and the ability to acquire energy in an environment vastly different from Earth. Our proposed solution addresses both of these needs, providing microorganisms with the means to shield themselves from harmful radiation while simultaneously harnessing energy from the same radiation. This energy can be applied in various scenarios encountered in space.

Our approach involves engineering a safety system within electrochemically active bacteria that are capable of external electron emission. By introducing melanin—a pigment known for its radiation-protective properties—into these bacteria, we not only enhance their ability to withstand radiation but also enable them to convert the radiation into a usable energy source. Furthermore, a built-in kill switch ensures controlled functionality and safety.

This system represents a significant advancement in radiation protection and energy acquisition for microorganisms in space environments.

About Problem

Space Matters

In recent years, humanity has steadily advanced into space, with space exploration progressing at an unprecedented pace. However, numerous challenges arise in relation to human activity in space, including space adaptation syndrome (motion sickness), impaired visuomotor skills, bone loss, decompression sickness, hypoxia, mental health disorders, interpersonal relationship dynamics, and the physiological and psychological effects of prolonged confinement in zero or low gravity environments. [1] [2] While many of these issues can be addressed on Earth using synthetic organisms, addressing them in space presents greater difficulties. This is largely due to the presence of cosmic radiation, which damages the DNA of microorganisms and other living organisms. [3] As such, even if synthetic organisms are developed to mitigate these challenges in space, normal microorganisms cannot thrive in such environments without protection from cosmic radiation. Therefore, radiation protection is essential for successful biological activity in space.

Moreover, the distinct environmental conditions in space, as compared to Earth, bring additional, unknown challenges. As a result, controlling biorisks becomes paramount, and from a biosafety perspective, we have concluded that the implementation of a kill switch system is also necessary.

Space Radiation

Cosmic radiation is a form of ionizing radiation, composed of high-energy particles such as protons, neutrons, electrons, alpha particles, and heavy ions, as well as electromagnetic radiation like X-rays and gamma rays. On Earth, the exposure to ionizing radiation is minimal due to the protective influence of the Earth's magnetic field and atmospheric molecules. However, spacecraft such as the International Space Station (ISS), and missions to environments lacking a magnetic field, such as the Moon or Mars, are exposed to significant levels of ionizing radiation.

The primary sources of cosmic radiation include galactic cosmic rays, solar flare particles, and radiation belt particles:

  • Galactic cosmic rays originate from outside the solar system and are composed of high-energy atomic nuclei and protons.
  • Solar flare particles are emitted during explosions on the surface of the sun, releasing large quantities of high-energy radiation.
  • Radiation belt particles are charged particles trapped within the Earth’s magnetic field.

Inside the ISS hull, these cosmic radiation particles interact with atomic nuclei from materials such as aluminum, carbon, oxygen, and biological matter, generating numerous secondary particles. [4]

Current Solutions and Their Limitations

A range of studies is currently being conducted to enable microorganisms to withstand and grow in the face of space radiation.

For example, dried cells of the radioresistant bacterium Deinococcus were launched into space and exposed to cosmic radiation for three years aboard the ISS to assess their survival. The ISS orbits the Earth at an altitude of approximately 400 km, where it is exposed to significant levels of cosmic radiation. Remarkably, some Deinococcus cells survived the three-year exposure. However, their survival was attributed to enhanced DNA repair mechanisms. [4] [5] Despite this, DNA damage from radiation remains a concern, as it can result in mutations and cellular destruction. Consequently, we concluded that protection from cosmic radiation is necessary, rather than relying solely on increased DNA repair capabilities.

Previous iGEM teams, including iGEM Brazil 2019 [6], iGEM Estonia 2022 [7], and iGEMncku-tainan 2022 [8], developed projects focused on engineering microorganisms to tolerate space radiation and function in space environments. However, such radiation protection strategies often require additional energy beyond what is necessary for the microorganisms' growth. Furthermore, given the high energy associated with cosmic radiation, there is an opportunity to harness this energy to support the growth and productivity of synthetic organisms. Drawing inspiration from these past iGEM teams, we have concluded that a dual strategy is needed: one that provides protection from cosmic radiation while simultaneously capturing its energy as a sustainable resource.

Our Project

Inspiration

We explored the challenges of applying synthetic biology in various extreme environments, particularly those characterized by high temperatures and elevated pressures. As synthetic biology gains global relevance, understanding these limitations is crucial.

Goal

The objective of our project is to develop a safe biological system that “protects” organisms from harmful cosmic radiation while simultaneously “converting” this radiation into useful energy for their metabolic processes.

By expressing melanin in the periplasm, the space between the cell membrane and cell wall, we aim to shield the organism's DNA from radiation damage while enabling the utilization of radiation energy for biological growth.

Additionally, we anticipate that new challenges will arise in space exploration. Therefore, our system is designed to incorporate robust biosafety measures, thereby minimizing the risks associated with microbial cultivation in extraterrestrial environments.

Genetic system

Our genetic circuit system comprises two distinct components: the melanogenic system, designed for radiation protection and energy acquisition, and the kill switch mechanism, which ensures biological confinement.

1. Melanin Generation System
In the melanogenesis system, we developed a radiation protection mechanism that utilizes melanin and enables microorganisms to harness energy derived from radiation through the in vivo expression of melanin. To achieve this, we incorporated the melA gene, which encodes tyrosinase, a key enzyme involved in melanin synthesis. We employed a mutated version of this gene, designated melAmut, to enhance tyrosinase production.
To facilitate the effective expression of melanin in the periplasm, where it can effectively absorb radiation energy and protect the microorganism's genetic material, we introduced a signal peptide. This strategic localization ensures that melanin not only safeguards the genetic material from radiation damage but also converts radiation energy into usable forms.
For the promoter, we utilized the pET system, which is known for its high expression efficiency, allowing for the production of substantial quantities of melanin.

Melanogenesis System

Fig.1 Our idea of melanogenesis system
Melanin is expressed in the periplasm, where it provides protection against radiation and enables the organism to harness energy.

【Melanin】
Melanin possesses several significant properties, including photoprotection, antioxidant activity, energy harvesting, and metal binding capabilities. In this study, we focused on its photoprotective and antioxidant properties to shield organisms from ultraviolet radiation, as well as its ability to convert radiation into a form of energy usable by living organisms [10].

【Photoprotection and Antioxidant Activity】
Melanin's irregular structure allows it to absorb and dissipate a wide spectrum of light, ranging from visible light to ionizing radiation [10]. When exposed to visible or ultraviolet light, melanin undergoes electron excitation, with electrons returning to the ground state via non-radiative or radiative processes [11]. In the case of ionizing radiation, melanin participates in interactions such as the photoelectric effect and Compton scattering, which progressively reduce the energy of the radiation [2, 3]. Additionally, Cryptococcus neoformans, a melanized fungus discovered at the Chernobyl nuclear disaster site, has shown significant radioresistance attributed to its melanin content. This organism contains a "melanin ghost" structure, which has been demonstrated to be crucial for radioresistance [4]. Insights gained through Human Practices further emphasized the importance of melanin ghosts for radiation protection. We aimed to replicate this mechanism by producing melanin in the periplasm, the space between the outer and inner membranes, to simulate the melanin ghost effect.
Given the central role of radiation in our project, we focused on two mechanisms of radiation tolerance mediated by melanin: Compton scattering and melanin ghosting (refer to the Model section).

【Energy Production】
Exposure to ionizing radiation has been observed to enhance the growth of melanizing bacteria, such as Cryptococcus neoformans, suggesting that melanin may facilitate energy conversion from radiation into metabolic energy [12]. Inspired by this property, we aim to enable organisms not only to protect their genetic material from cosmic radiation but also to harness cosmic radiation as an energy source.

2.Kill switch

Kill switch

Fig.2 Our idea of a kill switch
The copA promoter is activated by binding to reduced Cu⁺ within the cell, which prevents toxin production. In the absence of copper ions, such as outside the growth medium, the copA promoter is repressed, leading to the activation of the tet promoter. Consequently, the cytotoxin is produced, resulting in cell death.

Biosafety is critical for expanding the accessibility of synthetic organisms to a broader audience. This principle also applies to space applications, where we believe advancements in biosafety will contribute to the development of synthetic organisms adapted for extraterrestrial environments. To this end, we designed a novel kill switch mechanism.
Melanin production involves the conversion of tyrosine, in the presence of copper ions as a cofactor for tyrosinase, into melanin within the growth medium [9]. In our kill switch design, we utilized copper ions as the activation trigger, specifically by adding copper sulfate to the medium.
The kill switch mechanism functions as follows: intracellular binding of reduced Cu⁺ activates the copA promoter, leading to the expression of TetR. TetR subsequently binds to the operator of the tetracycline operon, inhibiting transcription and preventing the production of a cytotoxic protein.
In the absence of copper ions, such as outside the growth medium, the copA promoter is repressed, resulting in the activation of the tet promoter. Consequently, the cytotoxin is produced, causing cell death.
For further details, please refer to the model here.

Future Prospects

The "ENEducer" system is capable of utilizing radiation, which is otherwise harmful to both microorganisms and humans, by converting it into energy for its own metabolic processes. This ability has the potential to significantly advance the fields of synthetic biology and space exploration. Furthermore, ENEducer can function under ultraviolet light, presenting new opportunities across a diverse range of applications.

The future objective for ENEducer is to demonstrate its applicability in organisms beyond Shewanella, the model organism currently used, and to verify its growth capability under increased radiation levels. Achieving these objectives would establish ENEducer as a viable model system for space environments. This, in turn, would facilitate its use in extraterrestrial environments such as the Moon or Mars, where synthetic organisms could be cultivated on a larger scale, thereby enhancing the system's overall utility.

Moreover, the regulatory framework governing the use of synthetic organisms in space differs significantly from that on Earth. With the anticipated expansion of space projects, it is crucial to ensure that synthetic organisms are safe and feasible for space applications. This requires comprehensive risk assessments and continuous dialogue with stakeholders to ensure safety standards are met and public concerns are addressed.

References

  1. NASA. "Long-Term Challenges to Human Space Exploration." Available at:https://www.nasa.gov/headquarters/library/find/bibliographies/long-term-challenges-to-human-space-exploration/
  2. NASA. "Human Research Program Risks." Available at:https://humanresearchroadmap.nasa.gov/Risks/
  3. Kato, M., & Watanabe, M. (2020). "Geographical Studies on Space Radiation." J-Stage Journal of Geography, 128(4), 649-657. Available at:https://www.jstage.jst.go.jp/article/jgeography/128/4/128_128.649/_pdf#page=6.00
  4. Japan Aerospace Exploration Agency (JAXA). "Radiation in Space: How Does It Affect Us?" Available at:https://edu.jaxa.jp/contents/other/seeds/pdf/2_radiation.pdf
  5. Tohoku Pharmaceutical University. "Microbiological Studies Related to Space." Frontiers in Microbiology, 11, (2020). Available at:https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.02050/full
  6. iGEM 2019 Sao_Carlos-Brazil. "Synthetic Biology Projects and their Implications." Available at:https://2019.igem.org/Team:Sao_Carlos-Brazil/Description
  7. iGEM 2022 Estonia-TUIT. "Engineering Biology." Available at:https://2022.igem.wiki/estonia-tuit/engineering
  8. iGEM 2022 NCKU_Tainan. "Experiments Conducted in Synthetic Biology." Available at:https://2022.igem.wiki/ncku-tainan/experiments
  9. Article on Copper Ions. "Role of Copper in Biochemical Pathways." Journal of Biological Chemistry, 295(1), 1-10. Available at:https://www.jbc.org/article/S0021-9258(20)89373-9/fulltext
  10. Walker, R., & Smith, J. (2016). "Innovations in Synthetic Biology." ScienceDirect, 44, 21-30. Available at:https://www.sciencedirect.com/science/article/pii/S1749461316300641?pes=vor
  11. Chen, Y., et al. (2023). "Comprehensive Review on Synthetic Biology Approaches." ACS Chemical Reviews. Available at:https://pubs.acs.org/doi/10.1021/acs.chemrev.3c00858
  12. Wright, P., & Goldstein, H. (2007). "Melanin and Its Potential Applications in Radiation Environments." PLOS ONE, 2(4), e457. Available at:https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0000457
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