OBJECTIVES: THE GOAL OF THIS PROJECT IS TO USE ADVANCED MOLECULAR BIOLOGICAL TOOLS TO DISCOVER GENES IN A PHOTOSYNTHETIC MICROORGANISM THAT ARE CRITICAL FOR ADAPTATION TO A SPACEFLIGHT ENVIRONMENT. OUR OBJECTIVE IS TO COMPARE GROWTH ON THE ISS TO THAT OF EARTH AND ASSESS THE SURVIVABILITY AND ADAPTABILITY OF ONE OF EARTH'S MOST WIDE-SPREAD AND ROBUST PHOTOSYNTHETIC ORGANISMS CYANOBACTERIA. CYANOBACTERIA ARE PHOTOSYNTHETIC BACTERIA THAT FIX CARBON DIOXIDE AND GENERATE OXYGEN FROM SUNLIGHT AND HAVE BEEN EXPLOITED AS AN EFFICIENT BIOTECHNOLOGY PLATFORM FOR SYNTHESIS OF COMPLEX NATURAL PRODUCTS PROTEINS AND LIPIDS. THESE PROPERTIES MAKE THEM AN IDEAL CANDIDATE FOR SUPPORTING HUMAN SPACE EXPLORATION. HOWEVER FEW STUDIES HAVE BEEN DONE ASSESSING THEIR POTENTIAL FOR GROWTH IN SPACE. METHODOLOGY: WE WILL IDENTIFY GENES RELATED TO ADAPTION TO SPACEFLIGHT CONDITIONS USING AN EXISTING STATE-OF-THE-ART RANDOM-BARCODED TRANSPOSON (RB-TNSEQ) LIBRARY IN THE GENETIC MODEL CYANOBACTERIUM SYNECHOCOCCUS ELONGATUS (STRAIN PCC 7942). OUR LAB HAS DEVELOPED THIS GENOMIC MUTAGENESIS TOOL TO QUANTITATIVELY DETERMINE THE FITNESS VALUE OF EVERY GENE IN THIS ORGANISM UNDER A WIDE VARIETY OF SELECTIVE PRESSURES. THE EXPERIMENTAL DESIGN OF THE PROPOSED PROJECT IS TO GROW THE LIBRARY UNDER CONTROL AND GROUND-BASED CONDITIONS OF SIMULATED MICROGRAVITY AND SHORTENED PHOTOPERIOD AND THEN TO DEEP SEQUENCE THE BARCODES OF THE SURVIVING POPULATION TO DETERMINE THE FITNESS OF ALL GENES IN THE ORGANISM. BIOINFORMATICS IS USED TO CHARACTERIZE THE AFFECTED GENES AND METABOLIC PATHWAYS. THIS IS A POWERFUL METHOD TO IDENTIFY GENES PROVIDING SURVIVAL BENEFITS OR LIABILITIES IN SPACE. THESE EXPERIMENTS WILL GIVE US A GENOME-WIDE LOOK AT WHICH GENES ARE HELPFUL AND WHICH HARMFUL FOR ADAPTING TO SPACE. GENES IDENTIFIED DURING THE INITIAL SCREEN WILL BE SUBJECTED TO FURTHER SITE-SPECIFIC MUTATION AND ANALYSIS AND ENGINEERED MUTANTS WILL BE CONSTRUCTED FOR FURTHER TESTING TO ASSESS ADAPTATION TO SPACEFLIGHT. THESE STUDIES WILL BE SUPPORTED BY OUR CYANO-VECTOR GENE ASSEMBLY KIT WHICH ALLOWS FOR QUICK AND CUSTOMIZABLE GENE EXPRESSION STUDIES IN A DIVERSE SET OF CYANOBACTERIAL STRAINS. THIS PROJECT WILL PROVIDE A COMPREHENSIVE ANALYSIS OF GENES IMPORTANT DURING SPACEFLIGHT IN A PHOTOAUTOTROPH AND LAY THE FOUNDATION FOR GENETICALLY ENGINEERING ORGANISMS TO SURVIVE AND THRIVE DURING EXTRA-PLANETARY TRIPS. SIGNIFICANCE: THE PROPOSED PROJECT MEETS ROSBIO 2016 CALL PRIORITIES "1) EFFECTIVELY USE MICROGRAVITY AND OTHER SPACE ENVIRONMENT CHARACTERISTICS TO ENHANCE OUR UNDERSTANDING OF THE ADAPTATION AND FUNCTION OF BASIC BIOLOGICAL PROCESSES IN SPACEFLIGHT AND 2) DEVELOP A SCIENTIFIC AND TECHNOLOGICAL KNOWLEDGE BASE THAT WILL CONTRIBUTE TO A SAFE PRODUCTIVE HUMAN PRESENCE IN SPACE". THIS STUDY IS IDEAL FOR ELEMENT 3.4: MOLECULAR AND CELLULAR BIOLOGY FOCUS C: "INVESTIGATIONS THAT CHARACTERIZE THE ROLE OF A GENE OR A SET OF GENES IN AN ORGANISM'S ABILITY TO ACCLIMATE TO SPACEFLIGHT." THIS PROJECT USES CUTTING-EDGE MOLECULAR BIOLOGY TOOLS AND TECHNIQUES IN A MODEL PHOTOAUTOTROPH TO PERFORM AN IMPORTANT SCREEN OF ALL POSSIBLE GENE EFFECTS. PREVIOUS STUDIES ON A DIVERSE SET OF ORGANISMS HAVE SHOWN THAT SPACEFLIGHT AFFECTS CELLULAR OXIDATIVE STRESS RESPONSE PATHWAYS CELL WALL FORMATION AND CELL MORPHOLOGY. THIS PROJECT WILL HELP CREATE A ROADMAP FOR PURPOSEFUL GENETIC ENGINEERING TO GENERATE MORE ROBUST AND ADAPTABLE ORGANISMS. CYANOBACTERIA ARE AN UNDER-UTILIZED RESOURCE FOR SUPPORTING HUMAN SPACE EXPLORATION. THEY COULD SERVE AS A VITAL COMPONENT OF BIOLOGICAL LIFE SUPPORT SYSTEMS: GENERATING OXYGEN FROM CO2 H2O AND LIGHT; RECYCLING NUTRIENTS; AND SYNTHESIZING FOOD AND NUTRITIONAL COMPOUNDS SUCH AS OMEGA-3 FATTY ACIDS. THIS STUDY WOULD LAY THE FOUNDATION FOR THE USE AND ADAPTATION OF CYANOBACTERIA TO ADDRESS THE CHALLENGES LIVING ORGANISMS FACE IN SPACE.