Genetic expression by real-time PCR of bglA, bgl, CspA, gyrB, and 16S DNA genes from Shewanella sp. G5.

H.A. CRISTÓBAL; H. POMA; V. RAJAL; C. ABATE

Córdoba, Argentina

Simposio; XLIV Reunión Anual – Sociedad Argentina de Investigación en Bioquímica y Biología Molecula. IUBMB Symposium. Plant Biochemistry and Molecular Biology; 2008

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

ABSTRACT. Shewanella sp. G5 is a psychrotolerant Gram negative bacteria β-glucosidases (βGs) producers. This marine bacterium was isolate from the intestinal content of Munida subrrugosa in the Beagle Channel, Tierra del Fuego (Argentina); and grows between 4 and 37 ºC using cellobiose as carbon source. βGs are a heterogeneous group of enzymes with a broad substrate specificity range over different β-glucosides. The genes (bglA and bgl) that encoding of two βGs were characterized molecularly in previous studies. The primers design, RNA extraction, synthesis of cDNA and real time PCR assays using SYBRGreen were performed. The parameters assays to each condition were: temperature (10 and 30 ºC), carbon source (cellobiose and glucose) and culture media. bglA, bgl, CspA, gyrB and DNAr 16S genes were quantified and expressed as relative genetic expression from of each assays using 2-ΔΔCt method. Positive results were obtained for all genes during the optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. CspA, gyrB and DNAr 16S genes were quantified and expressed as relative genetic expression from of each assays using 2-ΔΔCt method. Positive results were obtained for all genes during the optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. time PCR assays using SYBRGreen were performed. The parameters assays to each condition were: temperature (10 and 30 ºC), carbon source (cellobiose and glucose) and culture media. bglA, bgl, CspA, gyrB and DNAr 16S genes were quantified and expressed as relative genetic expression from of each assays using 2-ΔΔCt method. Positive results were obtained for all genes during the optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. CspA, gyrB and DNAr 16S genes were quantified and expressed as relative genetic expression from of each assays using 2-ΔΔCt method. Positive results were obtained for all genes during the optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. over different β-glucosides. The genes (bglA and bgl) that encoding of two βGs were characterized molecularly in previous studies. The primers design, RNA extraction, synthesis of cDNA and real time PCR assays using SYBRGreen were performed. The parameters assays to each condition were: temperature (10 and 30 ºC), carbon source (cellobiose and glucose) and culture media. bglA, bgl, CspA, gyrB and DNAr 16S genes were quantified and expressed as relative genetic expression from of each assays using 2-ΔΔCt method. Positive results were obtained for all genes during the optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. CspA, gyrB and DNAr 16S genes were quantified and expressed as relative genetic expression from of each assays using 2-ΔΔCt method. Positive results were obtained for all genes during the optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. time PCR assays using SYBRGreen were performed. The parameters assays to each condition were: temperature (10 and 30 ºC), carbon source (cellobiose and glucose) and culture media. bglA, bgl, CspA, gyrB and DNAr 16S genes were quantified and expressed as relative genetic expression from of each assays using 2-ΔΔCt method. Positive results were obtained for all genes during the optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. CspA, gyrB and DNAr 16S genes were quantified and expressed as relative genetic expression from of each assays using 2-ΔΔCt method. Positive results were obtained for all genes during the optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. Beagle Channel, Tierra del Fuego (Argentina); and grows between 4 and 37 ºC using cellobiose as carbon source. βGs are a heterogeneous group of enzymes with a broad substrate specificity range over different β-glucosides. The genes (bglA and bgl) that encoding of two βGs were characterized molecularly in previous studies. The primers design, RNA extraction, synthesis of cDNA and real time PCR assays using SYBRGreen were performed. The parameters assays to each condition were: temperature (10 and 30 ºC), carbon source (cellobiose and glucose) and culture media. bglA, bgl, CspA, gyrB and DNAr 16S genes were quantified and expressed as relative genetic expression from of each assays using 2-ΔΔCt method. Positive results were obtained for all genes during the optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. CspA, gyrB and DNAr 16S genes were quantified and expressed as relative genetic expression from of each assays using 2-ΔΔCt method. Positive results were obtained for all genes during the optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. time PCR assays using SYBRGreen were performed. The parameters assays to each condition were: temperature (10 and 30 ºC), carbon source (cellobiose and glucose) and culture media. bglA, bgl, CspA, gyrB and DNAr 16S genes were quantified and expressed as relative genetic expression from of each assays using 2-ΔΔCt method. Positive results were obtained for all genes during the optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. CspA, gyrB and DNAr 16S genes were quantified and expressed as relative genetic expression from of each assays using 2-ΔΔCt method. Positive results were obtained for all genes during the optimization of the real time PCR. The amplification was verified by standard and dissociation curves analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method were carried out with the housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose. medium, 30 ºC and in presence of cellobiose. housekeeping genes (gyrB) also. The best values of relative genetic expression index for bglA and bgl were 27,67 and 30,17 respectively; and were obtained at the following assayed conditions: Brunner medium, 30 ºC and in presence of cellobiose.