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Construction of shuttle vectors for the genetic manipulation of diverse biotechnologically relevant budding yeasts (CROSBI ID 416846)

Ocjenski rad | diplomski rad

Arambašić, Kristian Construction of shuttle vectors for the genetic manipulation of diverse biotechnologically relevant budding yeasts / Štafa, Anamarija (mentor); Žunar, Bojan (neposredni voditelj). Zagreb, Prehrambeno-biotehnološki fakultet, . 2017

Podaci o odgovornosti

Arambašić, Kristian

Štafa, Anamarija

Žunar, Bojan

engleski

Construction of shuttle vectors for the genetic manipulation of diverse biotechnologically relevant budding yeasts

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.

codon optimization, yeast transformation, yeast integrative vectors, yeast replicating vectors

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Podaci o izdanju

37

30.06.2017.

obranjeno

Podaci o ustanovi koja je dodijelila akademski stupanj

Prehrambeno-biotehnološki fakultet

Zagreb

Povezanost rada

Biotehnologija