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Optimization of in vivo Cre/lox-Mediated Recombination in Chlamydomonas reinhardtii for Selectable Marker Free Recombinant Protein Production

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Abstract

The Cre/lox-mediated recombination system from the bacteriophage P1 is a fundamental genetic tool for the study of cell biology and genetics, as it allows for easy site-directed manipulation of genomes. Application of this system in the green unicellular model algae organism Chlamydomonas reinhardtii has not been sufficiently explored or applied in vivo. Establishing an efficient method of Cre/lox-mediated recombination will transform the way this organism can be studied and utilized as a bio-factory. A new reporter vector, pLox, was designed to easily screen for both cells with high protein expression—via mClover green fluorescent protein (GFP) detection before Cre recombinase activity—and cells with successful GFP deletion—via Oxyfluorfen herbicide resistance activation after Cre recombinase activity. Experiments were then designed to test if Cre/lox recombination is possible in vivo in C. reinhardtii using two approaches: 1) electroporating in exogenous Cre recombinase and 2) expressing Cre recombinase endogenously. In this thesis, the degrees to which the preliminary experiments and methods showed promise were documented. The design of pLox allows for straightforward verification of deletion, both through observing phenotypic change and PCR genotyping. Commercially supplied Cre recombinase was efficacious in recombining pLox in vitro, but our methods showed no positive results in vivo. However, one method of stably integrating our Cre recombinase expression cassette shows definite Cre excision, verified by genotyping and sequencing. In conclusion, in-vivo Cre/lox-mediated recombination in C. reinhardtii using our methods is promising, but Cre recombinase delivery must be optimized for efficiency and efficacy.

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This item is under embargo until September 10, 2023.