This proposal will utilize a combination of physiological and molecular studies to investigate proposal will utilize a combination of physiological and molecular studies to investigate methanotroph…

This federal contract award, NNX16AJ33G, was granted to The Leland Stanford Junior University (Stanford University) by the Shared Services Center, a civilian agency. The $327,000.00 grant supports research to investigate the production of the eukaryotic biomarker tetrahymanol by the methanotroph Methylomicrobium alcaliphilum under varying conditions of oxygen concentration, salinity, and temperature. The goal is to characterize the response of the tetrahymanol synthase gene and overall tetrahymanol production to these environmental factors. The research will also compare the impacts on a tetrahymanol synthase mutant strain. This grant does not have a set-aside designation, reflecting Stanford's status as a leading research institution. Stanford University is a renowned private university that frequently receives federal contract and grant awards across a wide range of scientific disciplines to support the government's research and development initiatives.

This proposal will utilize a combination of physiological and molecular studies to investigate proposal will utilize a combination of physiological and molecular studies to investigate methanotroph methylomicrobium alcaliphilum which we have shown produces a significant amount of tetrahymanol and varies the amount of this lipid IT produces under different environmental conditions. The goal is to identify whether oxygen concentration, salinity or temperature have an effect on tetrahymanol production. To do so, we will measure the following parameters transcription levels of the tetrahymanol synthase tetrahymanol production viability of a tetrahymanol synthase mutants morphological changes through electron microscopy all of these parameters will be measured under the following conditions oxygen supplied at 20%, 10%, 5%, or 1% 1 m, 0.5 m, 0.25 m, or 0.125 m sodium chloride growth temperature of 22C, 30C and 37C once we have identified conditions under which either transcription of tetrahymanol synthase or production of tetrahymanol is affected we will characterize the m. Alcaliphilum tetrahymanol synthase mutant THS under those conditions by measuring viability of the mutant through growth curves and colony forming unit plate assays changes in morphology in both the mutant and wild type through electron microscopy if we find mutant phenotypes under specific conditions, we will test other bacterial THS mutants including rhodopseudomonas palustris, an anoxygenic phototroph, and methylobacterium nodulans, a methylotrophic bacterium. The goal is to see if the correlation we find between environmental factors and THS expression and phenotypes extends beyond the aerobic methanotrophs.