Continuous in vitro evolution of a ribozyme ligase: a model experiment for the evolution of a biomolecule.
Evolution is a defining criterion of life and is central to understanding biological systems. However, the timescale of evolutionary shifts in phenotype limits most classroom evolution experiments to simple probability simulations. In vitro directed evolution (IVDE) frequently serves as a model system for the study of Darwinian evolution but produces noticeable phenotypic shifts in a matter of hours. An IVDE demonstration lab would serve to both directly demonstrate how Darwinian selection can act on a pool of variants and introduce students to an essential method of modern molecular biology. To produce an IVDE demonstration lab, continuous IVDE of a T500 ribozyme ligase population has been paired with a fluorescent strand displacement reporter system to visualize the selection of improved catalytic function. A ribozyme population is taken through rounds of isothermal amplification dependent on the self-ligation of a T7 promoter. As the population is selectively enriched with better ligase activity, the strand displacement system allows for the monitoring of the population's ligation rate. The strand displacement reporter system permits the detection of ligated ribozyme. Once ligated with the T7 promoter, the 5' end of the ribozyme displaces paired fluorophore-quencher oligonucleotides, in turn, generating visible signal upon UV light excitation. As the ligation rate of the population increases, due to the selection for faster ligating species, the fluorescent signal develops more rapidly. The pairing of the continuous isothermal system with the fluorescent reporting scheme allows any user, provided with minimal materials, to model the continuous directed evolution of a biomolecule.
Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas.
Michael P
MP
Ledbetter
Tony W
TW
Hwang
Gwendolyn M
GM
Stovall
Andrew D
AD
Ellington
D001483:Base Sequence; D055162:Biocatalysis; D019020:Directed Molecular Evolution / Q000295:instrumentation / Q000379:methods*; D005453:Fluorescence; D006801:Humans; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods; D008958:Models, Molecular; D008967:Molecular Biology / Q000193:education* / Q000379:methods; D008969:Molecular Sequence Data; D059370:RNA Folding; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012641:Selection, Genetic; D013334:Students; D013663:Teaching / Q000379:methods
D016337:RNA, Catalytic; D008025:Ligases
active learning; biochemical evolution; biotechnology education; laboratory exercises