Relative quantification of bgl-A and bgl genes in Shewanella sp. G5 by real time PCR

HÉCTOR A. CRISTÓBAL; RAMIRO POMA; VERÓNICA RAJAL; CARLOS ABATE

San Miguel de Tucumán

Congreso; Sociedad Argentina de Bioloía Molecular y Bioquímica (SAIB); 2009

SAIB

Shewanella sp. G5 was isolate from the intestinal content of Munida subrrugosa in the Beagle Channel (Argentina), and characterized as ß-glucosidases (ßGs) producers using cellobiose as carbon source inductor. ßGs (EC.3.2.1.21) are a heterogeneous group of enzymes classified into of the families 1 and 3 of the glycosyl hydrolases and their study present biotechnological interest. bglA and bgl genes which encoding two ßGs, were characterized molecularly in previous studies. The aim of this study was to determine the growth conditions where bgl-A and bgl increased its expression level . Shewanella sp. G5 was grown in different medium (LB and MMB), temperatures (10 and 30 ºC) and carbon sources (cellobiose and glucose). bgl-A, bgl and gyrB genes were quantified as relative genetic expression using 2-ΔΔCt method. The amplifications products were verified by standard and dissociation curves and amplot analysis (reporter of Ct). Normalization assays using 2-ΔΔCt method was carried out with gyrB housekeeping gene. The best values of 2–ΔΔCt to relative expression for bgl-A and bgl were 13, 14, 27 and 15, 19, 30 respectively; obtained in MMB incubated at 10 and 30 ºC using cellobiose as carbon source. Thus, these increased levels of expression in both genes allowed us to establish the optimum growth conditions for the best induction of ßGs in relation to other tested conditions.