Continuous in vitro Evolution of a Ribozyme Ligase: A Model Kit for The Evolution of a Biomolecule

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Michael Ledbetter, Tony W. Hwang, Gwendolyn M. Stovall and Andrew D. Ellington

The University of Texas at Austin, United States

There is a lack of hands-on demonstrations of Darwinian evolution in action in both high school and undergraduate level biology courses. This is likely due to the large time frames needed to observe the changes caused by the forces of evolution. In vitro directed evolution (IVDE) closely mimics natural evolution but produces observable phenotypic changes in a matter of hours as opposed to years. An IVDE demo kit would serve to both directly demonstrate the forces of Darwinian evolution and introduce students to an essential method of modern synthetic biology. In order to produce an IVDE demo kit, continuous IVDE of a T500 ribozyme ligase based pool has been paired with a fluorescence-based strand displacement reporter system in order to visualize the evolution of improved catalytic function. Ribozyme pools are taken through rounds of isothermal-based amplification dependent on the self-ligation of a T7 promoter. Dilution between rounds of evolution will select for ribozymes with faster ligation kinetics. As the pool evolves the strand displacement system allows for the monitoring of the pool’s ligation rate. The strand displacement reporter system allows for visual detection of ligated ribozyme. When ligated with the T7 promoter, the 5’ end of the ribozyme possesses enough complementarity with a fluorophore-labeled DNA oligo to displace, through toehold mediated strand displacement, an quencher-labeled DNA oligo, in turn, generating a visible signal upon UV light excitation. As the ligation rate of the pool increases due to the selection for faster ligating species, the student will observe more rapid development of fluorescent signal in later rounds of evolution. The pairing of the continuous isothermal system with the fluorescence-based strand displacement detection scheme allows any user, provided with minimal materials to model the continuous directed evolution of a biomolecule.