Cholesterol bioconversion by Tetrahymena thermophila

MARIA BELEN DE LUCA; CLARA NUDEL; RODRIGO H GONZÁLEZ; ALEJANDRO D. NUSBLAT

Camerino

Conferencia; Conference on Ciliate Molecular Biology 2015; 2015

Tetrahymena in the Classroom   Cholesterol bioconversion by Tetrahymena thermophila Maria Belen De Luca1, Clara B. Nudel1, Rodrigo H. Gonzalez1 and Alejandro D. Nusblat1 1 Instituto de Nanobiotecnología NANOBIOTEC, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, (C1113AAD) Buenos Aires, Argentina   ?Design and optimization of biological systems? is an elective course for undergraduate students to complete the career of biochemistry at the school of Biochemistry of the University of Buenos Aires in Buenos Aires, Argentina. The topics of the course are: Introduction to the isolation and selection of microorganisms of industrial interest. Breeding methods and systems for cloning and expression. Regulation and coordination of microbial metabolism. Introdution to Systems and Synthetic Biology and its methodological tools. Composition, formulation, design and optimization of culture media. Biotransformations of industrial interest. Study of processes related to the production of food, drugs, environmental biotechnology and biofuels.   In this course we use Tetrahymena thermophila as an example of a biological system for the biotransformation of metabolites of industrial interest. We focus on sterol metabolism, specifically in  the conversion of cholesterol to pro-vitamin D3. The workshop lasts five days of 4 hours each and this year (2015) 14 students between 22 and 25 years old have been enrolled. They were grouped in 3-4 people?s teams. The students use wild type strains to measure the conversion of cholesterol to pro-vitamin D3. They also use different recombinant strains (knock out) to learn how sterol pathway could be modify and obtain diverse sterols moieties. The students take samples at different times (0, 24, 48 and 72 hs) of bioconversion and they identify and measure the sterols concentration by HPLC. They also identified by genomic PCR, the strain of use.    Additionally, the students compare the biotransformation rate, growing the ciliate in plates and in 1L fermentator. The measurement of the cell density is performed by Neubauer Chamber count. Finally, the students use a fluorescence microscope to see both the substrate and the involved enzymes localization. To this purpose, we use fluorescent Bodipy-cholesterol and GFP-fusion sterol desaturases strains. The last day, each team has to make an oral presentation, explaining all the results obtained and fulfill a series of selected questions to approve the workshop.