Project Grant 2221721

SBIR PHASE I: BROADBAND FOCUSING FOR NON-INVASIVE CELL METABOLOMICS -THE BROADER IMPACT OF THIS SMALL BUSINESS INNOVATION RESEARCH (SBIR) PHASE I PROJECT IS AIMED TOWARD ADVANCING SUSTAINABLE PRODUCTION OF CHEMICALS USING SYNTHETIC BIOLOGY. HERE, SINGLE-CELL MICROBES ARE ENGINEERED TO PRODUCE VALUABLE METABOLITES USING ENZYMES RATHER THAN SOURCING THESE CHEMICALS FROM PETROLEUM. DEVELOPING GENETICALLY ENGINEERED CELL STRAINS WITH HIGH YIELD REMAINS AN ONGOING EFFORT DUE TO THE COMPLEXITIES IN HOW GENETIC CODE LEADS TO PHENOTYPE EXPRESSION. THIS PROBLEM IS ADDRESSED USING A BOTTOM-UP APPROACH TO SCREEN MICROBE POPULATIONS AT THE SINGLE-CELL LEVEL. THE METHOD DEPLOYED TO IDENTIFY METABOLITE CONTENT IN INDIVIDUAL CELLS IS BASED ON INFRARED (IR)-ABSORPTION SPECTROSCOPY WHICH IS LABEL-FREE, QUANTITATIVE, AND NON-DESTRUCTIVE. SYNTHETIC BIOLOGY IS POISED TO DISRUPT THE CHEMICAL VALUE-CHAIN BY PROVIDING AN ALTERNATIVE TO PETROLEUM-BASED CHEMICALS THAT IS SUSTAINABLE AND CARBON-NEUTRAL. ONCE A HIGHLY PRODUCTIVE CELL IS IDENTIFIED IT CAN BE SELECTIVELY PROPAGATED TO CREATE ENRICHED CELL LINES. INNOVATIONS IN OPTICAL MICROSCOPY ARE REQUIRED TO IMPROVE THE PERFORMANCE OF THE CELL SCREENING INSTRUMENTS, WHICH WILL ALLOW HIGH-RESOLUTION FOCUSING ACROSS A BROAD SPECTRAL RANGE. THE UPGRADED PLATFORM WILL OPTIMIZE YIELD MORE QUICKLY, PROVIDING VALUE BY REDUCING THE UPFRONT COST TO DEVELOP NEW INDUSTRIAL CELL STRAINS. THE PROPOSED PROJECT EMPHASIZES OPTICAL ENGINEERING TO DEVELOP A MICROSCOPE DESIGNED FOR HIGH-RESOLUTION CHEMICAL IMAGING BASED ON MOLECULAR VIBRATIONAL IR-ABSORPTIONS. THIS IS ACHIEVED BY DEPLOYING FOCUSING ELEMENTS THAT OPERATE OVER A BROAD SPECTRAL RANGE THAT EXTEND STANDARD OPTICAL MICROSCOPES TO INCLUDE MID-INFRARED LIGHT SOURCES. THE OPTICAL INSTRUMENT WILL BE USED TO EVALUATE CHEMICAL CONTENT IN INDUSTRIAL MICROBE STRAINS AND DEVELOP ENRICHED CELL LINES. THESE SINGLE-CELL MICROBE POPULATIONS ARE ENGINEERED TO PRODUCE ENZYMES USED TO CATALYZE THE SYNTHESIS OF VALUABLE METABOLITES. YIELDS FROM INDIVIDUAL CELLS, HOWEVER, ARE VARIABLE DUE TO GENETIC MUTATIONS IN THE POPULATION. THEREFORE, A QUANTITATIVE ANALYTICAL TOOL BASED ON IR-SPECTROSCOPY THAT CAN NON-DESTRUCTIVELY IDENTIFY HIGHLY PRODUCTIVE CELLS FOR SELECTIVE PROPAGATION IS EXTREMELY DESIRABLE. THIS BOTTOM-UP APPROACH FOR METABOLOMIC CELL SCREENING AND DIRECTED EVOLUTION IS AN INNOVATION AS IT IS LABEL-FREE, NON-INVASIVE, AND HAS STRONG CHEMICAL SPECIFICITY. IN THIS PROJECT, THE TEAM WILL STUDY AN INDUSTRIAL MICROALGAE STRAIN USED AS A LOW-COST FEEDSTOCK SUPPLEMENT AND IDENTIFY CELLS RICH IN PROTEIN CONTENT TO ENHANCE THE OVERALL PROTEIN YIELD. THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.