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Javdi M.,Institute of Industrial Biotechnology | Haghnazari A.,University of Zanjan | Tohidfar M.,Agricultural Biotechnology Research Institute of Iran | Ghareyazi B.,Agricultural Biotechnology Research Institute of Iran
Euphytica | Year: 2010

Successful identification of homozygous and heterozygous transgenic plant with currently available techniques such as southern blot hybridization, dot blot hybridization, fluorescence in situ hybridization (FISH) and so on, demands tedious and time-consuming procedures with a high proportion of ambiguous results. Real-time PCR is a quantitative and extremely precise method with high throughput that could be applied to the analysis of large number of plants differing only by a factor of two in the amount of target sequences. In the present study, we determined zygosity level of transgenes in cotton [Gossypium hirsutum L.] with two zygosity assays, based on TaqMan technology that uses a fluorogenic probe which hybridizes to a PCR target sequence flanked by primers. TPS, a single copy gene per haploid Gossypium hirsutum genome was used as the endogenous reference to estimate copy number of transgene. Both assays were accurate and reproducible in determination of the number of transgenes present in a cell line. These methods are standard curves and Delta delta Ct method. © 2009 Springer Science+Business Media B.V.

Abdullah R.,Lahore College for Women University | Ul-Haq I.,Institute of Industrial Biotechnology
Natural Product Research | Year: 2015

α-Amylase produced by a mutant strain of Aspergillus oryzae EMS-18 has been purified to homogeneity as judged by sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified by using 70% ammonium sulphate precipitation followed by anion exchange chromatography on DEAE-Sephadex column and gel filtration on Sephadex G-100. An enzyme purification factor of 9.5-fold was achieved with a final specific activity of 1987.7 U/mg protein and overall yield of 23.8%. The molecular weight of purified α-amylase was estimated to be 48 kDa by SDS-PAGE. The purified enzyme revealed an optimum assay temperature and pH 40°C and 5.0, respectively. Except Ca++ all other metal ions such as Mg, Mn, Na, Zn, Ni, Fe, Cu, Co and Ba were found to be inhibitory to enzyme activity. © 2015 Taylor and Francis.

Javaid M.M.,Institute of Industrial biotechnology | Ikram-Ul-Haq,Institute of Industrial biotechnology | Sohail M.I.,Institute of Industrial biotechnology | Bokhari S.A.I.,Institute of Industrial biotechnology
Pakistan Journal of Botany | Year: 2012

Two bacterial cultures of Corynebacterium glutamicum and Brevibacterium flavum were screened for maximum Llysine synthesis in shake flask. Among these, the best culture i.e., B. flavum was subjected to mutagenesis through UV irradiation, ethidium bromide and nitrous acid treatment for enhanced production of L-lysine. Among five isolated mutants screened for efficiency towards L-lysine synthesis B. flavum IIBUV2 was found to be the best producer. B. flavum IIBUV2 and its wild type were, then, compared to find out an appropriate cultivation media and conditions. Maximum L-lysine production of 8.8 g/l was obtained when mutant was cultivated on medium containing (%, w/v): glucose 3.0, peptone 0.5, NH4SO4 0.1 and K2HPO2 0.005, pH 7.5 at 32°C with 8% inoculum. When the mutant was cultivated in 7.5 L stirred fermenter the lysine yield was increase up to 17.8 g/l after 120h at 300 rpm agitation and 4.0 VVM aeration.

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