Results for “whole-genome sequencing”

NASA NTRS2017

Identification of Novel Desiccation-Tolerant S. cerevisiae Strains for Deep Space Biosensors

NASA's BioSentinel mission, a secondary payload that will fly on the Space Launch System's first Exploration Mission (EM-1), utilizes the budding yeast S. cerevisiae to study the biological response to the deep space radiation environment. Yeast samples are desiccated prior to launch to suspend growth and metabolism while the spacecraft travels to its target heliocentric orbit beyond Low Earth Orbit. Each sample is then rehydrated at the desired time points to reactivate the cells. A major risk in this mission is the loss of cell viability that occurs in the recovery period following the desiccation and rehydration process. Cell survival is essential for the detection of the biological response to features in the deep space environment, including ionizing radiation. The aim of this study is to mitigate viable cell loss in future biosensors by identifying mutations and genes that confer tolerance to desiccation stress in rad51, a radiation-sensitive yeast strain. We initiated a screen for desiccation-tolerance after rehydrating cells that were desiccated for three years, and selected various clones exhibiting robust growth. To verify retention of radiation sensitivity in the isolated clones - a crucial feature for a successful biosensor - we exposed them to ionizing radiation. Finally, to elucidate the genetic and molecular bases for observed desiccation-tolerance, we will perform whole-genome sequencing of those rad51 clones that exhibit both robust growth and radiation sensitivity following desiccation. The identification and characterization of desiccation-tolerant strains will allow us to engineer a biological model that will be resilient in face of the challenges of the deep space environment, and will thus ensure the experimental success of future biosensor missions.

S. cerevisiae↗

NASA NTRS2017

Biosentinel: Improving Desiccation Tolerance of Yeast Biosensors for Deep-Space Missions

BioSentinel is one of 13 secondary payloads to be deployed on Exploration Mission 1 (EM-1) in 2019. We will use the budding yeast Saccharomyces cerevisiae as a biosensor to determine how deep-space radiation affects living organisms and to potentially quantify radiation levels through radiation damage analysis. Radiation can damage DNA through double strand breaks (DSBs), which can normally be repaired by homologous recombination. Two yeast strains will be air-dried and stored in microfluidic cards within the payload: a wild-type control strain and a radiation sensitive rad51 mutant that is deficient in DSB repairs. Throughout the mission, the microfluidic cards will be rehydrated with growth medium and an indicator dye. Growth rates of each strain will be measured through LED detection of the reduction of the indicator dye, which correlates with DNA repair and the amount of radiation damage accumulated. Results from BioSentinel will be compared to analog experiments on the ISS and on Earth. It is well known that desiccation can damage yeast cells and decrease viability over time. We performed a screen for desiccation-tolerant rad51 strains. We selected 20 re-isolates of rad51 and ran a weekly screen for desiccation-tolerant mutants for five weeks. Our data shows that viability decreases over time, confirming previous research findings. Isolates L2, L5 and L14 indicate desiccation tolerance and are candidates for whole-genome sequencing. More time is needed to determine whether a specific strain is truly desiccation tolerant. Furthermore, we conducted an intracellular trehalose assay to test how intracellular trehalose concentrations affect or protect the mutant strains against desiccation stress. S. cerevisiae cell and reagent concentrations from a previously established intracellular trehalose protocol did not yield significant absorbance measurements, so we tested varying cell and reagent concentrations and determined proper concentrations for successful protocol use.

BioSentinel↗

NASA NTRS2017

Identification of Novel Desiccation-Tolerant S. cerevisiae Strains for Deep Space Biosensors

NASA's BioSentinel mission, a secondary payload that will fly on the Space Launch Systems first Exploration Mission (EM-1), utilizes the budding yeast S. cerevisiae to study the biological response to the deep space radiation environment. Yeast samples are desiccated prior to launch to suspend growth and metabolism while the spacecraft travels to its target heliocentric orbit beyond Low Earth Orbit. Each sample is then rehydrated at the desired time points to reactivate the cells. A major risk in this mission is the loss of cell viability that occurs in the recovery period following the desiccation and rehydration process. Cell survival is essential for the detection of the biological response to features in the deep space environment, including ionizing radiation.The aim of this study is to mitigate viable cell loss in future biosensors by identifying mutations and genes that confer tolerance to desiccation stress in rad51, a radiation-sensitive yeast strain. We initiated a screen for desiccation-tolerance after rehydrating cells that were desiccated for three years, and selected various clones exhibiting robust growth. To verify retention of radiation sensitivity in the isolated clonesa crucial feature for a successful biosensorwe exposed them to ionizing radiation. Finally, to elucidate the genetic and molecular bases for observed desiccation-tolerance, we will perform whole-genome sequencing of those rad51 clones that exhibit both robust growth and radiation sensitivity following desiccation. The identification and characterization of desiccation-tolerant strains will allow us to engineer a biological model that will be resilient in face of the challenges of the deep space environment, and will thus ensure the experimental success of future biosensor missions.

S. cerevisiae↗

NASA NTRS2017

Biosentinel: Improving Desiccation Tolerance of Yeast Biosensors for Deep-Space Missions

BioSentinel is one of 13 secondary payloads to be deployed on Exploration Mission 1 (EM-1) in 2019. We will use the budding yeast Saccharomyces cerevisiae as a biosensor to determine how deep-space radiation affects living organisms and to potentially quantify radiation levels through radiation damage analysis. Radiation can damage DNA through double strand breaks (DSBs), which can normally be repaired by homologous recombination. Two yeast strains will be air-dried and stored in microfluidic cards within the payload: a wild-type control strain and a radiation sensitive rad51 mutant that is deficient in DSB repairs. Throughout the mission, the microfluidic cards will be rehydrated with growth medium and an indicator dye. Growth rates of each strain will be measured through LED detection of the reduction of the indicator dye, which correlates with DNA repair and the amount of radiation damage accumulated. Results from BioSentinel will be compared to analog experiments on the ISS and on Earth. It is well known that desiccation can damage yeast cells and decrease viability over time. We performed a screen for desiccation-tolerant rad51 strains. We selected 20 re-isolates of rad51 and ran a weekly screen for desiccation-tolerant mutants for five weeks. Our data shows that viability decreases over time, confirming previous research findings. Isolates L2, L5 and L14 indicate desiccation tolerance and are candidates for whole-genome sequencing. More time is needed to determine whether a specific strain is truly desiccation tolerant. Furthermore, we conducted an intracellular trehalose assay to test how intracellular trehalose concentrations affect or protect the mutant strains against desiccation stress. S. cerevisiae cell and reagent concentrations from a previously established intracellular trehalose protocol did not yield significant absorbance measurements, so we tested varying cell and reagent concentrations and determined proper concentrations for successful protocol use.

BioSentinel↗

NASA NTRS2005

Genome-wide transcriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes

The important role that cilia and flagella play in human disease creates an urgent need to identify genes involved in ciliary assembly and function. The strong and specific induction of flagellar-coding genes during flagellar regeneration in Chlamydomonas reinhardtii suggests that transcriptional profiling of such cells would reveal new flagella-related genes. We have conducted a genome-wide analysis of RNA transcript levels during flagellar regeneration in Chlamydomonas by using maskless photolithography method-produced DNA oligonucleotide microarrays with unique probe sequences for all exons of the 19,803 predicted genes. This analysis represents previously uncharacterized whole-genome transcriptional activity profiling study in this important model organism. Analysis of strongly induced genes reveals a large set of known flagellar components and also identifies a number of important disease-related proteins as being involved with cilia and flagella, including the zebrafish polycystic kidney genes Qilin, Reptin, and Pontin, as well as the testis-expressed tubby-like protein TULP2.

Polycystic Kidney Diseases/genetics↗