Vývoj fluorescenční reportérové linie \kur{Babesia divergens}

Šárka BORSODI

Bachelor's thesis

Abstract:

B. divergens, a model species of this project, is an agent of economically important bovine babesiosis and life-threatening human babesiosis primarily found in Europe. Despite its importance, key biological processes underlying B. divergens parasitism in the host cell are still not well understood, partly due to the absence of B. divergens-specific gene-targeting techniques. Therefore, we aim to establish a "gold standard" genetic tool and develop B. divergens fluorescent reporter lineage. In this thesis, we set up the necessary background for the construction of episomal B.divergenes-specific GFP plasmid that will be further used for the parasite in vivo imaging. We identified and sequenced B.divergens-specific 5' and 3'untranslated regions that will serve for genes-of-interest expression in the desired plasmid. As a selective drug, we will employ blasticidin (BSD) that successfully inhibited the B. divergens in vitro multiplication. Effective inhibitory BSD concentrations were determined using the newly established and optimized technique of parasitemia determination by fluorescent DNA labelling analysed by flow cytometry. Using obtained data, we designed the episomal GFP-expressing plasmid and proposed consecutive steps of its construction using the Gibson assembly strategy that we successfully verified by in silico cloning.

Language used: Czech

Date on which the thesis was submitted / produced: 9. 12. 2021

Thesis defence

• Supervisor: RNDr. Marie Jalovecká, Ph.D.

UNIVERSITY OF SOUTH BOHEMIA IN ČESKÉ BUDĚJOVICE

Faculty of Science

Bachelor programme / field:
Biology / Biology