AS HUMAN SPACE EXPLORATION EXPANDS BEYOND LOWER EARTH ORBIT IT IS NECESSARY TO CHARACTERIZE THE EFFECTS OF SPACE RADIATION MICROGRAVITY AND THE COMBINATION THEREOF ON CELLS. BECAUSE IT IS COMPLICATED TO HAVE LARGE SAMPLE NUMBERS WHEN STUDYING THE EFFECTS OF DIFFERENT FACTORS ON HUMANS SCIENTISTS COMMONLY USE MODEL ORGANISMS THAT SHARE SOME OF THE KEY ASPECTS BEING STUDIED. IN THIS CASE WE WILL USE YEAST AS AROUND 70% OF ITS ESSENTIAL GENES HAVE A SIGNIFICANT HUMAN HOMOLOG. MORE SPECIFICALLY THIS PROJECT WILL USE A MOLECULARLY BARCODED YEAST GENOME-WIDE KNOCKDOWN COLLECTION THAT WILL ENABLE THE SYSTEMATIC INTERROGATION OF THE EFFECT OF MICROGRAVITY SPACE RADIATION AND A COMBINATION THEREOF IN EACH GENE. EACH STRAIN IN THE COLLECTION HAS A SINGLE GENE DELETED AND A REPRESENTATIVE MOLECULAR BARCODE THAT ENABLES QUANTIFYING THE FITNESS OF EACH MUTANT UNDER THE TEST CONDITIONS BY MEASURING THE RELATIVE ABUNDANCE AT DIFFERENT POINTS IN TIME. TO DIFFERENTIATE THE EFFECTS OF MICROGRAVITY AND SPACE RADIATION ON EACH STRAIN AN EXPERIMENTAL SET WILL BE FLOWN BEYOND THE VAN ALLEN BELTS ON ORION S EXPLORATION MISSION 1 (EM-1) (CONSIDERED IN MICROGRAVITY AND IRRADIATED BY SPACE RADIATION) AND EQUIVALENT SETS WILL BE CULTURED ASYNCHRONOUSLY ON BOARD THE INTERNATIONAL SPACE STATION (ISS) (CONSIDERED IN MICROGRAVITY BUT MOSTLY ALTHOUGH NOT COMPLETELY PROTECTED OF SPACE RADIATION BY THE VAN ALLEN BELTS) IN OUR SMART INCUBATOR (SPACE AUTOMATED BIOPRODUCT LAB (SABL)) AND ON EARTH (ALSO IN A GROUND SABL). EACH OF THE ISS AND EARTH EXPERIMENTS WILL INCLUDE TWO SETS: ONE WHERE THE TEMPERATURE PROFILE EXPERIENCED DURING THE EM-1 FLIGHT IS REPLICATED AND A SECOND CULTURED AT A CONSTANT TEMPERATURE TO DETERMINE THE POTENTIAL ROLE OF TEMPERATURE VARIATION ON THE RESULTS FROM EM-1. THE FIRST AIM OF THIS PROJECT IS TO IDENTIFY THE METABOLIC AND GENOMIC PATHWAYS IN YEAST AFFECTED BY MICROGRAVITY SPACE RADIATION AND A COMBINATION OF BOTH. THE SECOND ONE IS TO DIFFERENTIATE BETWEEN GRAVITY AND RADIATION EXPOSURE ON SINGLE-GENE DELETION MUTANTS ABILITY TO THRIVE IN THE SPACEFLIGHT ENVIRONMENT. WE HYPOTHESIZE THAT MUTANTS LACKING GENES ASSOCIATED WITH DNA REPAIR RECOMBINATION AND REPLICATION WILL HAVE LOWER SURVIVABILITY RATES BEYOND THE VAN ALLEN BELTS THAN THEIR BELOW VAN ALLEN BELTS- OR EARTH-CONTROLS THE EXPERIMENT IS DESIGNED TO HAVE A CONTROLLED START AFTER ORION IS PAST THE VAN ALLEN BELTS GROW ~21 GENERATIONS OF THE DELETION SERIES AND FIX OR PRESERVE SAMPLES FOR POST-FLIGHT ANALYSES. SHOULD THE AUTOMATED CONTROLLED APPROACH BE CONSIDER INAPPROPRIATE FOR IMPLEMENTATION ON EM-1 WE HAVE A PASSIVE APPROACH THAT IS BASED ON DOTTING EACH MUTANT INDIVIDUALLY ON AGAR. WE HAVE PERFORMED BOTH APPROACHES IN SPACE IN THE PAST. THIS PROJECT WILL ADDRESS THREE SPACE BIOLOGY PROGRAM SCIENCE ELEMENTS THREE OBJECTIVES THREE GUIDING QUESTIONS AND FOUR DECADAL SURVEY S HIGHEST PRIORITY RECOMMENDATIONS BY PRESERVING NUCLEIC ACIDS OF DIFFERENT GENERATIONS OF THE YEAST DELETION SERIES CULTURES GROWN IN SPACE BEYOND AS WELL AS BELOW THE VAN ALLEN BELTS (AND UPLOADING THE GENOMIC AND TRANSCRIPTOMIC DATA TO GENELAB).