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Doctoral Thesis
DOI
https://doi.org/10.11606/T.11.2019.tde-20191220-124312
Document
Author
Full name
Luiz Humberto Gomes
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Piracicaba, 2000
Supervisor
Title in Portuguese
Construção de um vetor com o gene GFP de Aequorea victoria para a transformação de Saccharomyces cerevisiae
Keywords in Portuguese
CONTAMINAÇÃO
EXPRESSÃO GÊNICA
FERMENTAÇÃO INDUSTRIAL
LEVEDURAS
MARCADOR MOLECULAR
PROTEÍNA VERDE FLUORESCENTE
VETOR PLASMIDIAL
Abstract in Portuguese
Diversos métodos tem sido propostos para a identificação de leveduras contaminantes em processos fermentativos que utilizam leveduras da espécie Saccharomyces cerevisiae, mas nenhum deles oferece resultados absolutamente satisfatórios com a exatidão e precisão necessárias. Considerando que a identificação poderia ser facilitada com o uso de genes mercadores, foi construído um vetor plasmidial pYGFP3 com o gene da GFP (“green fluorescent protein”) extraída da Aequorea victoria, sob o controle do promotor de ADH2 (álcool desidrogenase II) de Saccharomyces cerevisiae o qual apresenta repressão por glicose. O vetor pYGFP3 foi utilizado para transformação de leveduras S. cerevisiae HD 93.15 D (MAT a, his 3, leu2, trpl, ura3) e X2904-3C (MATa, met1, trp1, ura3), que expressam o gene GFP3 somente em condições de baixo teor de glicose, não interferindo com outras funções metabólicas ou dispêndio de energia durante o processo fermentativo. Repicações sucessívas demonstraram a estabilidade do gene marcador nos transformantes, indicando que o método é viável para identificação simples e segura desta linhagem, podendo-se constituir em recurso adicional de identificação da levedura frente a outras contaminantes do processo fermentativo
Title in English
Construction of a vector carrying the GFP gene from Aequorea victoria to transforme Saccharomyces cerevisiae
Abstract in English
Several methods have been proposed for identify spoilage yeast during fermentation processes, that uses Saccharomyces cerevisiae, therefore none of them offers absolutely acurancy and precision needed. Considering that the identification could be facilitate with the use of gene markers, a plasmid vector was constructed carrying the GFP gene (green fluorescent protein) from Aequorea victoria, under the control of ADH2 (alcohol dehydrogenase II) promoter from Saccharomyces cerevisiae, which is repressed by glucose. The p YGFP3 vector was used to transform yeast Saccharomyces cerevisiae HD 93.15D (MAT a, his 3, leu2, trpl, ura3) and X2904-3C (MATa, metl, trp 1, ura3) and the GFP3 was expressed only under low levels of glucose, not interfering in other metabolic functions of the cell neither spending energy during the fermentative process. Successive replications shows gene stability into the transformed cells, indicating its viability for simple and secure of this strain, being considered an additional tool to identify spoilage yeast in fermentative processes.
 
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GomesLuizHumberto.pdf (5.28 Mbytes)
Publishing Date
2019-12-20
 
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