Task Progress:
|
This project is developing engineered yeast to be assayed for performance in extreme deep-space environments using the BioSensor microfluidics system that was developed for the BioSentinel CubeSat as part of Artemis I. The BioSensor uses light emitting diodes (LED) at three wavelengths to allow measurements of cell growth and color changes in yeast cultures. The project research activities complement and directly support a NASA Payloads and Research Investigations on the Surface of the Moon project entitled, “Lunar Explorer Instrument for space biology Applications (LEIA).” The LEIA suite of instruments includes the BioSensor, a charged particle, linear energy transfer spectrometer, and fast neutron detector, which will be delivered to the lunar surface as part of a Commercial Lunar Payload Services (CLPS) mission. The two radiation detectors will allow direct correlation between radiation exposure with yeast growth and production in the BioSensor.
This project has made significance progress on all four specific aims as follows.
Aim 1) Develop methods to predict synthetic biology production traits, namely carotenoids and recombinant proteins, using multivariate statistical models based on three wavelength light absorbance.
We determined that optical density measurements at 465 nm and 850 nm can be used to detect beta-carotene production in microtiter plate formats. To measure protein content, we engineered baker’s yeast to express Enhanced Cyan-Green Fluorescent Protein (ECGFP). Although in year 1 of the project we found that florescence of ECGFP was predictive of ECGFP levels in stationary cultures, absorbance at 465 nm is not significant enough to detect ECGFP expression. These experiments provided evidence that the spectroscopy approaches planned for carotenoid detection in LEIA BioSensor experiments are feasible. Additional experiments with desiccated yeast strains are ongoing and will be used to calibrate 465 nm absorbance to beta-carotene production levels.
Aim 2) Engineer carotenoid-expressing yeast strains that enhance sensitivity or resistance to expected lunar surface environment stressors.
We generated twelve yeast strains that are expected to alter sensitivity to abiotic stresses, such as reactive oxygen species (ROS) or radiation. Four strains were confirmed to be more sensitive to long-term storage of dry cells in ambient, laboratory conditions. Five of the yeast strains tested expression of damage suppressor (Dsup) or cytoplasmic abundant heat soluble (CAHS) genes from tardigrades, which have been shown to enhance radiation or desiccation tolerance in plants, animals, or yeast.
3) Engineer a non-conventional yeast species to express a blue light compatible marker for synthetic biology-enabled production.
We developed a single-locus beta-carotene biosynthesis pathway construct that can be integrated into multiple yeast species. The first iteration of this construct produced low levels of beta-carotene in three baker’s yeast strains. We are currently revising the construct to improve beta-carotene production for eventual transfer into Crabtree-negative yeast.
4) Use BioSensor microfluidics cards to test inoculum, desiccation, and spectroscopy methods to measure cell growth and product formation.
We completed initial tests with BioSensor microfluidics cards in ground support equipment (GSE). These tests showed feasibility to detect beta-carotene production using visible light absorbance at 465 nm and Near-Infrared Spectroscopy (NIR) absorbance at 850 nm.
|
|
Abstracts for Journals and Proceedings
|
Liddell LC, NN, Chau JW, Raj CG, Gentry D, Settles AM, Santa Maria SR. "Adapting a flexible BioSensor Platform for biological studies on the Moon." 39th Annual Meeting of the American Society for Gravitational and Space Research, Washington, DC, November 13-18, 2023. Abstracts. 39th Annual Meeting of the American Society for Gravitational and Space Research, Washington, DC, November 13-18, 2023. , Nov-2023
|
|
Abstracts for Journals and Proceedings
|
Raj CG, Ball NN, Chau JW, Gentry D, Gilbert R, Liddell LC, Settles AM, Santa Maria SR. "Microbial optical data processing: A key step in the metabolic assessment of Lunar Explorer Instrument for Space Biology Applications (LEIA) and Biosentinel’s payload data." 39th Annual Meeting of the American Society for Gravitational and Space Research, Washington, DC, November 13-18, 2023. Abstracts. 39th Annual Meeting of the American Society for Gravitational and Space Research, Washington, DC, November 13-18, 2023. , Nov-2023
|
|
Abstracts for Journals and Proceedings
|
Settles AM, Ball NN, Broddrick JT, Chau JW, Ehresmann B, Gentry DM, Acevedo JG, Heffern LE, Hindupur A, James LM, Lee JA, Liddell LC, Timucin LR, Vu ST, Pletcher DL, Hassler DM, Santa Maria SR. "LEIA: An investigation of radiation risks to biology at the lunar South Pole." 39th Annual Meeting of the American Society for Gravitational and Space Research, Washington, DC, November 13-18, 2023. Abstracts. 39th Annual Meeting of the American Society for Gravitational and Space Research, Washington, DC, November 13-18, 2023. , Nov-2023
|
|