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Phenol stress response in Corynebacterium glutamicum
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Abstract(s)
O principal objetivo deste trabalho foi estudar o efeito dos fatores Sigma A, Sigma C, Sigma E, Sigma H e um controlo (sem fator sigma) no promotor de Corynebacterium glutamicum.
Procedeu-se à preparação dos promotores ParoF, Pcg1277, PcydA e PctaB, por associação de primers (PAROFPEPR Forward e PAROFPEPR Reverse, PCG1277PEPR Forward e PCG1277PEPR Reverse, PCYDAPEPR Forward e PCYDAPEPR Reverse, PCTABPEPR Forward e PCTABPEPR Reverse). Após este passo, seguiu-se a ligação, transformação e isolamento dos fragmentos obtidos. Seguidamente, foram preparadas as células competentes de C. glutamicum, em que os fatores sigma utilizados foram, Sigma A, Sigma C, Sigma E, Sigma H e uma estirpe sem fator sigma para funcionar como fator de controlo. Foi realizada a transformação em que se obtém o sistema de dois plasmídeos (pEC-XT99A e pEPR1) com os promotores construídos com a associação de primers e as estirpes com os diversos fatores sigma. Todas as transformações e preparações de células foram confirmadas por eletroforese em gel de agarose.
Iniciaram-se os ensaios de fluorescência, que foram divididos em duas partes: no primeiro ensaio o meio de cultura continha apenas antibióticos, e no segundo ensaio houve adição de fenol ao meio de cultura. Em ambas as situações, existem estirpes com e sem IPTG, com e sem fenol, e com fenol e IPTG.
No primeiro ensaio, confirmou-se que a presença dos promotores provoca uma alteração na expressão dos fatores sigma. Nomeadamente, a presença do promotor ParoF incita uma sobre expressão do Sigma H; o promotor Pcg1277 provoca uma sobre expressão do Sigma E e os promotores PcydA e PctaB, ambos incitam que o Sigma C tenha uma expressão acentuada.
No segundo ensaio, com a adição de fenol, não houve diferenças nos fatores sigma que têm uma sobre-expressão devido à presença de determinados promotores, pois embora o fenol crie um ambiente tóxico na célula, com a presença de fenol houve expressão dos fatores sigma.
The main goal of this project was to study the effect of the sigma factors Sigma A, Sigma C, Sigma E and Sigma H, and a control (no sigma) on the promoters of C. glutamicum. It was performed the preparation of the promoters ParoF, Pcg1277, PcydA and PctaB, with the re-association of primers (PAROFPEPR Forward and PAROFPEPR Reverse, PCG1277PEPR Forward and PCG1277PEPR Reverse, PCYDAPEPR Forward and PCYDAPEPR Reverse, PCTABPEPR Forward and PCTABPEPR Reverse). After this step, ligation, transformation and isolation of the fragments were made. Then, competent cells of Corynebacterium glutamicum were prepared and the sigma factors used were Sigma A, Sigma C, Sigma E, Sigma H and a control sigma to use as a control. The two-plasmid strain was obtained by transformation by electroporation, and all the steps that include transformation and preparation were confirmed by PCR and then electrophoresis in agarose gel. The fluorescence assays were initiated, and they were divided in two concepts: in the first assay the medium had only the antibiotics and the second assay contained phenol. In both situations, there were strains with and without IPTG, with and without phenol, and strains with both. IPTG functions by binding to the lacI repressor and altering its conformation, which prevents the repression of the β-galactosidase coding gene lacZ. In the first set of assays, it was confirmed that the presence of the promoters stimulates the overexpression of the sigma factors. Namely, ParoF makes Sigma H have a higher expression, Pcg1277 does the same to Sigma E and both PcydA and PctaB have the same effect on Sigma C. In the second set of assays, with the addition of phenol, there were no changes in terms of which sigma has a higher overexpression to certain promoters. However, phenol is a toxic element to the cell, but there still was some expression in the strains that had phenol.
The main goal of this project was to study the effect of the sigma factors Sigma A, Sigma C, Sigma E and Sigma H, and a control (no sigma) on the promoters of C. glutamicum. It was performed the preparation of the promoters ParoF, Pcg1277, PcydA and PctaB, with the re-association of primers (PAROFPEPR Forward and PAROFPEPR Reverse, PCG1277PEPR Forward and PCG1277PEPR Reverse, PCYDAPEPR Forward and PCYDAPEPR Reverse, PCTABPEPR Forward and PCTABPEPR Reverse). After this step, ligation, transformation and isolation of the fragments were made. Then, competent cells of Corynebacterium glutamicum were prepared and the sigma factors used were Sigma A, Sigma C, Sigma E, Sigma H and a control sigma to use as a control. The two-plasmid strain was obtained by transformation by electroporation, and all the steps that include transformation and preparation were confirmed by PCR and then electrophoresis in agarose gel. The fluorescence assays were initiated, and they were divided in two concepts: in the first assay the medium had only the antibiotics and the second assay contained phenol. In both situations, there were strains with and without IPTG, with and without phenol, and strains with both. IPTG functions by binding to the lacI repressor and altering its conformation, which prevents the repression of the β-galactosidase coding gene lacZ. In the first set of assays, it was confirmed that the presence of the promoters stimulates the overexpression of the sigma factors. Namely, ParoF makes Sigma H have a higher expression, Pcg1277 does the same to Sigma E and both PcydA and PctaB have the same effect on Sigma C. In the second set of assays, with the addition of phenol, there were no changes in terms of which sigma has a higher overexpression to certain promoters. However, phenol is a toxic element to the cell, but there still was some expression in the strains that had phenol.
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Keywords
Fatores sigma Sobre expressão Fluorescência Promotores IPTG Fenol Sigma factors Overexpression Fluorescence Promoters Phenol