Abstract EANA2025-101

This work is part of the HZ2022-funded ALCYONE project (Autonomous Living Cell analYsis ON-chip for Evaluation of space Environment Effects), which aims to develop innovative low-power integrated lab-on-chip biosensors for assessing radiation-induced damage in living organisms during nanosatellite missions. The primary objective is to engineer bacterial and cyanobacterial biosensors capable of real-time monitoring of the combined effects of space radiation and altered gravity on microorganisms. The biosensors are based on the bacterium Escherichia coli MG1655, known for its sensitivity to radiation and desiccation, alongside the cyanobacterium Chroococcidiopsis sp. CCMEE 029, a robust extremophile exhibiting remarkable tolerance to desiccation and ionizing radiation. These microbial models provide a comprehensive framework
for investigating biological responses to the complex stressors of the space environment. The development strategy focuses on constructing a luciferase-based genetic reporter system using shuttle plasmids designed for stable maintenance following extended desiccation. Ground-based simulations serve as a proof of concept, involving exposure to ionizing radiation under altered gravity conditions. These experiments are conducted using a Rotary Cell Culture System (Synthecon), integrated within an irradiation facility, to mimic the multifactorial conditions encountered in space. This setup enables real-time monitoring of gene expression dynamics under increasing radiation doses, simulating the environmental stressors expected during prolonged deep-space missions, such as crewed travel to Mars or extended orbital habitation.
Results
Since space radiation mainly causes oxidative stress and DNA damage, an in-silico analysis of the genome of Chroococcidiopsis sp. CCMEE 029 was performed using a bioinformatic approach to identify genes involved in the SOS response regulated by LexA and RecA. RT-qPCR experiments confirmed the overexpression of recA and lexA genes following oxidative stress induced by hydrogen peroxide, validating recA as a suitable target for stress sensing. Based on these results, a synthetic plasmid was engineered, incorporating the luciferase sequence under the control of the recA promoter from Chroococcidiopsis to activate luciferase gene expression in response to DNA
damage. The bacterial biosensor based on E. coli was rigorously tested for plasmid stability during desiccation: it retained plasmid integrity after 7 and 28 days in a desiccated state when resuspended in 100 mM D-trehalose. Furthermore, luciferase expression was significantly induced over time following exposure to varying doses of UV-C irradiation, confirming the biosensor’s responsiveness to radiation-induced stress. Similarly, the cyanobacterial biosensor based on Chroococcidiopsis was evaluated after exposure to increasing UV-C doses, demonstrating its ability to detect and respond to high levels of radiation stress. These findings highlight the robustness and sensitivity of both microbial biosensors for monitoring space-like stress conditions. Chemiluminescence assays, conducted using a “Tecan M-Plex” plate reader, validated the real- time monitoring capabilities of the luciferase reporter system in both models.
Conclusion
This research contributes to the development of autonomous biosensing platforms for continuous, real-time biological monitoring during space missions. The integration of such systems into nanosatellite platforms may significantly enhance life-support technologies, improve astronaut safety, and deepen our understanding of microbial responses to space environmental conditions.