Naslov
Konstrukcija plazmidinih vektora za genetičku manipulaciju različitih biotehnološki značajnih pupajućih kvasaca
(Construction of shuttle vectors for the genetic manipulation of diverse biotechnologically relevant budding yeasts)

Autori
Arambašić, Kristian

Vrsta, podvrsta i kategorija rada
Ocjenski radovi, diplomski rad, diplomski

Fakultet
Prehrambeno-biotehnološki fakultet

Mjesto
Zagreb

Datum
30.06

Godina
2017

Stranica
37

Mentor
Štafa, Anamarija

Neposredni voditelj
Žunar, Bojan

Ključne riječi
optimizacija kodona, transformacija kvasca, kvaščevi integrativni plazmidi, kvaščevi replikativni plazmidi
(codon optimization, yeast transformation, yeast integrative vectors, yeast replicating vectors)

Sažetak
Model yeast Saccharomyces cerevisiae is used in both basic and applied research, mostly because it is amenable to genetic manipulation. Using extensive collection of selective markers and shuttle vectors, it is possible to delete or modify several genes at the same time and to introduce new metabolic pathways. However, due to easy manipulation of its genome, yeast S. cerevisiae is an exception among biotechnologically interesting (budding) yeast species, although many of them have properties that could be used in particular bioprocesses. For many yeast species, replication origins are unknown while for others, although identified, they are not widely used. Moreover, some yeast species employ non-standard genetic code that renders standard selective markers ineffective. Thus, it is needed to first construct a vector and to confirm its functionality, which wastes both time and resources. To streamline genetic engineering of non-Saccharomyces species, we codon optimised four common antibiotic markers, obtaining genes functional across the clade of budding yeasts, regardless of the particular species’ genetic code. These markers were used to construct a set of four integrating plasmids and twenty shuttle vectors, each combining one marker with one of five diverse replication origins. Using these plasmids, we successfully transformed five divergent budding yeast species that shared last common ancestor more than 250 million years ago. Furthermore, we determined antibiotic concentrations needed for the selection of transformants and developed a single transformation protocol for those five species. Finally, we constructed a broad spectrum yeast shuttle vector which contains all five replication origins. Vectors constructed here should greatly facilitate genetic manipulation of diverse budding yeast species and, consequently, encourage their application in biotechnology.

Izvorni jezik
Engleski

Znanstvena područja
Biotehnologija

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