Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker

Background: A commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele. Results: A K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth. Conclusions: In this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii

A review of the systematic biology of fossil and living bony-tongue fishes, Osteoglossomorpha (Actinopterygii: Teleostei)

The bony-tongue fishes, Osteoglossomorpha, have been the focus of a great deal of morphological, systematic, and evolutionary study, due in part to their basal position among extant teleostean fishes. This group includes the mooneyes (Hiodontidae), knifefishes (Notopteridae), the abu (Gymnarchidae), elephantfishes (Mormyridae), arawanas and pirarucu (Osteoglossidae), and the African butterfly fish (Pantodontidae). This morphologically heterogeneous group also has a long and diverse fossil record, including taxa from all continents and both freshwater and marine deposits. The phylogenetic relationships among most extant osteoglossomorph families are widely agreed upon. However, there is still much to discover about the systematic biology of these fishes, particularly with regard to the phylogenetic affinities of several fossil taxa, within Mormyridae, and the position of Pantodon. In this paper we review the state of knowledge for osteoglossomorph fishes. We first provide an overview of the diversity of Osteoglossomorpha, and then discuss studies of the phylogeny of Osteoglossomorpha from both morphological and molecular perspectives, as well as biogeographic analyses of the group. Finally, we offer our perspectives on future needs for research on the systematic biology of Osteoglossomorpha

Algumas aprendizagens construídas durante a brincadeira de pipa: o que está em jogo Some learning constructed during kite flying: what is at stake

Como recorte de pesquisa assentada em objeto lúdico tradicional, mapeamos algumas atividades eliciadas pela brincadeira de pipa, focalizando as aprendizagens informais e os brinquedos-ponte como forma privilegiada de transmissão cultural. Potentes em sua capacidade de aglutinação, esses brinquedos encontram traduções singulares para cada brincante, com significativo impacto em sua bagagem vivencial, por colocarem em jogo aprendizagens de corpo inteiro, cotidianas e situadas, emergentes da articulação entre pessoas, objetos e outros elementos que se afetam mutuamente na produção de efeitos. Utilizando a Teoria Ator-Rede como suporte teórico-metodológico, seguimos grupos de brincantes durante as temporadas dos anos de 2005 e 2006 e realizamos entrevistas com pessoas que tiveram a pipa como objeto privilegiado de suas infâncias, tomando os fragmentos desse material para operar misturas entre textos acadêmicos e textos não-acadêmicos.<br>As part of the research on about a traditional playful object, we tried to register some kinds of learning produced by kite flying, focusing on informal learning, as well as bridge-toys as a privileged transmission. Powerful in its capacity of joining people of different ages together, these toys can represent singular translations for each player with meaningful impact in their life experience once they place full body quotidian learning at stake, emergent from the articulation between people, objects and other elements that affect one another mutually in the production of effects. Using the ActorNetwork Theory as the theoretical methodological support, we followed some playing groups during the 2005 and 2006 flying kite seasons and interviewed people who had kite as a privileged object in their childhood. This material's fragment was mixed with academic and non academic texts