 |
Abstract EANA2024-27 |
|
“Development of bacterial and cyanobacterial biosensors for real-time monitoring of space radiation response in microgravity conditions”
Introduction
The project part of the HZ2022 funded project ALCYONE: Autonomous Living Cell analYsis ONchip for Evaluation of space Environment Effects: low-power integrated lab-on-chip for the assessment of radiation damage on living systems in nanosatellite missions.
The main objective of the project is to design biosensors using the bacterium Escherichia coli MG1655 and the cyanobacterium Chroococcidiopsis sp. CCMEE 029 for monitoring the effect of space radiation and microgravity on microorganisms. Chroococcidiopsis sp. CCMEE 029 is a suitable candidate since it is desiccation-, and radiation-tolerant in contrast to E. coli MG1655 which is radiation-, desiccation-sensitive bacterium.
The development approach will be based on the design of a genetic system for a luciferase-based reporter system by using shuttle plasmids reported to be maintained after desiccation. As a proof of-concept suitable promoters will be used in ground-based simulations (Rotary Cell Culture System, Synthecon inside a ionizing radiation facility) in order to expose the developed biosensors to increasing doses of ionizing radiation in microgravity conditions, up to that expected after a prolonged permanence into deep space (e.g. travel to Mars) and evaluate the by real-time monitored of selected genes with the luciferase reporter system.
Preliminary results
Since space radiations cause DNA damage through the production of oxidative stress, an in-silico analysis of the genome of Chroococcidiopsis sp. CCMEE 029 was started by using a bioinformatic approach to identify genes involved in the SOS response which are under the regulation of LexA & recA. Preliminary results obtained by RT-qPCR showed in Chroococcidiopsis sp. CCMEE 029 the over expression of the recA gene after treatment with H2O2. In order to use the recA promoter sequence of Chroococcidiopsis sp. CCMEE 029 a synthetic derivate of plasmid pQE_30UA, named “pQE_30UA_recA_029_S2_luc_pDU1” was obtained.
To first validate the system, chemiluminescence analyses were conducted in E. coli MG1655 and Chroococcidiopsis sp. CCMEE 029 and detected via "ChemiDoc Imaging System" and "Tecan" instrument.
REFERENCES:
[1] Billi, D. Plasmid stability in dried cells of the desert cyanobacterium Chroococcidiopsis and its potential for GFP imaging of survivors on Earth and in space. Origins of Life and Evolution of Biospheres, 42, 235-245 (2012).
[2] Billi, D., Friedmann, E. I., Helm, R. F., & Potts, M. Gene transfer to the desiccation-tolerant cyanobacterium Chroococcidiopsis. Journal of Bacteriology, 183(7), 2298-2305 (2001).
[3] Billi, D., Staibano, C., Verseux, C., Fagliarone, C., Mosca, C., Baqué, M., ... & Rettberg, P. Dried biofilms of desert strains of Chroococcidiopsis survived prolonged exposure to space and Mars-like conditions in low Earth orbit. Astrobiology, 19(8), 1008-1017 (2019).
[4] Billi, D., Verseux, C., Fagliarone, C., Napoli, A., Baqué, M., & de Vera, J. P. A desert cyanobacterium under simulated Mars-like conditions in low Earth orbit: implications for the habitability of Mars. Astrobiology, 19(2), 158-169 (2019).
[5] Cottin, H., Kotler, J. M., Billi, D., Cockell, C., Demets, R., Ehrenfreund, P., ... & Klamm, B. A. Space as a tool for astrobiology: review and recommendations for experimentations in Earth orbit and beyond. Space Science Reviews, 209, 83-181 (2017).
[6] Napoli, A., Micheletti, D., Pindo, M., Larger, S., Cestaro, A., De Vera, J. P., & Billi, D. Absence of increased genomic variants in the cyanobacterium Chroococcidiopsis exposed to Mars like conditions outside the space station. Scientific Reports, 12(1), 8437 (2022).