BACKGROUND: THE CENTRAL DOGMA IS QUINTESSENTIAL TO LIFE ON EARTH ENABLING LIVING SYSTEMS TO CARRY OUT INFORMATION PROCESSING METABOLISM REPRODUCTION AND HOMEOSTASIS. RNA A UNIQUELY INFORMATION-DENSE AND CATALYTIC BIOMOLECULE SITS AT THE CENTER OF THIS REQUIREMENT FOR LIFE IN ALL KNOWN BIOLOGICAL SYSTEMS AND MIGHT HAVE BEEN RESPONSIBLE FOR THE TRANSITION FROM SIMPLE CHEMICAL TO COMPLEX BIOLOGICAL SYSTEMS. AMONG RNA COMPONENTS DEFINING THE CENTRAL DOGMA THERE IS PERHAPS NONE MORE IMPORTANT THAN THE RIBOSOME WHICH SERVES AS A CELLULAR NEXUS OF INFORMATION TRANSFER RESOURCE ALLOCATION AND GROWTH MODULATION. CONSERVATION OF THE RIBOSOME IS USED TO TRACE EVOLUTIONARY TRAJECTORIES ACROSS THE TREE OF LIFE FROM LUCA YET THERE IS LITTLE EVIDENCE TO SUPPORT RRNA-BASED PHYLOGENETIC RECONSTRUCTIONS OF INTERMEDIATE VERSIONS OF THE TRANSLATIONAL APPARATUS. METHODOLOGIES THAT ENABLE THE RECONSTRUCTION AND EXPERIMENTAL VALIDATION OF ANCESTRAL RIBOSOME INTERMEDIATES ALONG PLAUSIBLE EVOLUTIONARY TRAJECTORIES TO ITS CURRENT FORM WOULD TRANSFORM OUR UNDERSTANDING OF THE ORIGIN OF LIFE ON EARTH ELUCIDATE THE EVOLUTION OF MODERN RIBOSOMES AND POTENTIALLY INFORM ROUTES FOR TRANSLATION ELSEWHERE IN THE COSMOS. MAJOR GOALS: HISTORICALLY ORIGINS-OF-LIFE RESEARCHERS HAVE FOCUSED ON ABOTTOM-UP APPROACH TO THE PREDICTION OF INTERMEDIATE PRE-BIOTIC BIOMOLECULES IN WHICH ARTIFICIAL CHEMICAL SYSTEMS ARE CONSTRUCTED IN ATTEMPTS TO RECREATE CONDITIONS ON THE EARLY EARTH. CONVERSELY ATOP-DOWN APPROACH IN WHICH EXISTING BIOLOGICAL SYSTEMS ARE REDUCED TO THEIR UNDERLYING COMPONENTS WOULD INFORM THE FUNCTIONAL AND MINIMAL COMPONENTS THAT MAY HAVE EXISTED IN A PREBIOTIC ERA. WE PROPOSE TO TAKE A TOP-DOWN APPROACH TO THE MINIMIZATION OF THE RIBOSOME WITHIN THE FUNCTIONALITY-RELEVANT ENVIRONMENT OF THE CELL. SUCH A COMBINATION OF THEORETICAL AND EXPERIMENTAL ANALYSIS WOULD INFORM THE CHEMICAL FUNCTIONALITY DEFINING TRANSLATION. HOWEVER STUDIES AIMED AT PROBING CELLULAR TRANSLATION HAVE RARELY ACCESSED SUCH A DETAILED UNDERSTANDING OF THE MINIMAL REQUIREMENTS FOR TRANSLATION. THIS IS A DIRECT CONSEQUENCE OF DIFFICULTIES ASSOCIATED WITH RIBOSOME MANIPULATION IN VIVO AS TRANSLATION CANNOT BE EASILY UNCOUPLED FROM CELLULAR VIABILITY OR OVERALL FITNESS. THUS OUR PROPOSED WORK REQUIRES THE REMOVAL OF CONFOUNDING ATTRIBUTES REGULATING CELLULAR VIABILITY THAT MAY BE INCORRECTLY ASSUMED TO INFLUENCE THE REQUIREMENTS FOR THIS BIOMOLECULAR ACTIVITY. SPECIFIC AIMS: TO ACHIEVE THESE GOALS WE PROPOSE TO INTEGRATE SYNTHETIC BIOLOGY COMPUTATIONAL AND PHYLOGENETIC RRNA ANALYSIS AND EVOLUTIONARY PRINCIPLES TO INFORM PLAUSIBLE AND TESTABLE MINIMAL RIBOSOME INTERMEDIATES EN ROUTE TO THE EXTANT TRANSLATION MACHINERY OF THE CELL: 1. AIM I: CONSTRUCTION OF A DEFINED SENSOR THAT RECAPITULATES RIBOSOMAL ACTIVITY IN E. COLI AS A MODULAR AND ORTHOGONAL PLATFORM BY UNCOUPLING TRANSLATION FROM HOST VIABILITY. 2. AIM II: APPLICATION OF THIS SENSOR PLATFORM TOWARDS THE IN VIVO ANALYSIS OF THE EFFECTS OF RRNA TRUNCATIONS AND/OR MUTATIONS ON TRANSLATION OF A SINGULAR SYNTHETIC MRNA TRANSCRIPT. 3. AIM III: OPTIMIZATION OF MINIMIZED RRNA VARIANTS USING PHAGE-ASSISTED CONTINUOUS EVOLUTION (PACE) TO REVEAL COMPENSATORY MUTATIONS THAT ENHANCE ACTIVITY. CONCLUSION: OUR PROPOSED WORK AIMS TO OVERCOME CHALLENGES ASSOCIATED WITH RIBOSOMAL MANIPULATION IN VIVO AND PROVIDES AN INNOVATIVE FRAMEWORK FOR THE DERIVATION OF MINIMIZED RIBOSOMES. WHILE THIS IS AN AMBITIOUS INTEGRATION OF EVOLUTIONARY PRINCIPLES AND SYNTHETIC BIOLOGY TO ILLUMINATE THE ORIGINS OF TRANSLATION WE HOPE THAT IT WILL SUPPLEMENT THE EXISTING (BUT INCOMPLETE) MOLECULAR RECORD OF RIBOSOMAL EVOLUTION AND PROVIDE NOVEL INSIGHT INTO POTENTIAL PROTO-RIBOSOMAL GENOTYPES AND PHENOTYPES. OUR PROPOSED WORK WOULD MARK THE FIRST MAJOR EFFORT TO MINIMIZE THIS COMPLEX MACROMOLECULAR MACHINE PROVIDING EXPERIMENTAL EVIDENCE FOR A POTENTIAL ROUTE TOWARDS THE DEVELOPMENT OF LIFE OF EARTH.