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Optimizacija metod za visokozmogljivo transformacijo kvasovkr Saccharomyces cerevisiae
ID Hafner, Luka (Author), ID Petrovič, Uroš (Mentor) More about this mentor... This link opens in a new window

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Abstract
Kemijsko transformacijo kvasovke Saccharomyces cerevisiae običajno izvajamo v mikrocentrifugirkah. Kadar je potrebno izvesti veliko transformacij, kot na primer pri pripravi knjižnice sevov z izbitimi geni, to postane zamudno. Tu se pojavi potreba po visoko zmogljivi metodi za transformacijo. V diplomski nalogi smo želeli optimizirati postopek, ki bi prihranil čas pri takšnih nalogah. Opazovali smo rast kvasovke S. cerevisiae na mikrotitrski plošči s 96 vdolbinicami in pripravili rastne krivulje. Prilagodili smo protokol za transformacijo na mikrotitrski plošči na osnovi protokola za transformacijo v mikrocentrifugirkah. S prilagojenim protokolom smo transformirali kvasovke S. cerevisiae s plazmidnimi in linearnima vektorjema. Ugotovili smo, da je transformacija celic v zgodnji eksponentni fazi rasti vsaj 10-krat bolj učinkovita od transformacije celic v pozni eksponentni fazi rasti. Kot pomembna se je izkazala natančna in enakomerna redčitev prekonočnih kultur, da hkrati dosežejo zgodnjo eksponentno fazo rasti. Transformacija na mikrotitski plošči je obetavna metoda za visokozmogljivo transformacijo kvasovke S. cerevisiae, ki omogoča znaten prihranek časa. Nadaljnje optimizacije, kot so uporaba mikrotitrskih plošč z ukrivljenim dnom in spreminjanje časa toplotnega šoka, lahko prispevajo k izboljšanju učinkovitosti transformacije.

Language:Slovenian
Keywords:Saccharomyces cerevisiae, transformacija, rastna krivulja
Work type:Bachelor thesis/paper
Typology:2.11 - Undergraduate Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Year:2023
PID:20.500.12556/RUL-149331 This link opens in a new window
COBISS.SI-ID:168254979 This link opens in a new window
Publication date in RUL:06.09.2023
Views:258
Downloads:24
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Secondary language

Language:English
Title:Optimization of a high-throughput yeast transformation method for Saccharomyces cerevisiae
Abstract:
Chemical transformation of the yeast Saccharomyces cerevisiae is typically performed in microcentrifuge tubes. However, when a large number of transformations needs to be carried out, such as in the preparation of knockout strain libraries, it becomes time-consuming. Therefore, there is a need for a high-throughput method of transformation. In this thesis, we aimed to develop a procedure that would save time in such tasks. We observed the growth of S. cerevisiae on a 96-well microtiter plate and prepared growth curves. We adapted the transformation protocol based on microcentrifuge tubes for transformation in the wells of a microtiter plate. Using the modified protocol, we transformed S. cerevisiae using plasmids and linear vectors. We found that the transformation of cells in the early exponential growth phase is at least 10 times more efficient than transformation of cells in the late exponential growth phase. Uniform dilution of overnight cultures appeared to be crucial for the diluted cultures to simultaneously achieve early exponential growth phase. Transformation on a microtiter plate is a promising method for high-throughput transformation of S. cerevisiae, enabling significant time savings. Further optimizations, such as using microtiter plates with a curved bottom and adjusting the heat shock time, may contribute to improving the efficiency of transformation.

Keywords:Saccharomyces cerevisiae, transformation, growth curve

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