[Enhansment of Biodesulfurization activity via the over expression of the oxidoreductase gene from R.erytheropolis IGTS8 using pet 21 a vector]

Biological desulforezation is a microbial procedure which uses manipulated bacterial strains for increasing of desulforezation from petroleum products. The method is being tested by several big oil companies for making a safe fuel and for preventing air pollution one of the methods for enhancement of desulforezation of microbial oil strains is increasing of copy number of oxidoreductase gene, this gene is able to supply the required FMNH2 for desulforezation genes of Rhodococcus erythropolis IGTS8, which is used as a model micro organism in desulforezation researches, was amplified with PCR and was cloned in to the PTZ57R vector. Then the oxidoreductase gene was extracted by agarose gel DNA extraction kit as a 600 bp fragment and was inserted in to the pkk223-3 expression vector. After induction, the desired protein was weakly detected as a 25 KD band in SDS-PAGE gel. However using another expression vector [PET 21a], a sharp band was detected in the same area. At present, the studies was focused on increasing the production in National Instutute of genetic Engineering and bio technology we are trying to apply genetic Engineering tools to increase gene dosage of oxidoreductase gene to enhancement of desulforezation gene expression

Cloning of glucose oxidase gene in PKK233-3

Glucose oxidase [GO] has found a variety of industrial applications such as food, chemical and personal care industries. However one of the most important application of GO is used as diagnostic kits. The aim of study was isolation of GO gene from a recombinant vector [PET21aGO] and sub cloning and expression in PKK233-3 vector. Recombinant PET21a GO was extracted from E.coli DH5alpha and was digested with Restriction Enzymes; BamHI, Hindlll then isolated GO gene [1.8kb] and cloned in PKK233-3. PKK233-3GO was transformed in to E.coli DH5alpha. Our data demonstrated that the GO gene has expected size in agarose gel electrophoresis and also the cloned Go has a correct size after restriction analysis. The GO gene was cloned in prokaryotic host. This is a report of cloning of GO gene in Iran that can be used for further cloning of that gene in expression vectors for production of recombinant Enzyme

Detection and cloning of desulfurization operon sox [dsz A and B] in rhodococcus FMF native bacterium

Native Rhodococcus FMF bacterium was selected among several isolated Iranian bacterium. Primary studies have shown that the bacterium use dibenzothiophene as a sulfur source. Since desulfurization is taken by a biochemical pathway, the present project was designed to detect, clone and sequence of desulfurization genes of the bacterium. Desulfurization operon was isolated from Rhodococcus erythropolis IGTS8 and was used as a probe for detection of desulfurization 4S pathway genes by southern blotting technique. Specific primers were designed and the dsz A and B genes were amplified by polymerase chain reaction [PCR]. PCR products were recovered from agarose gel and were cloned into the PTZ57R vector. In this study, recombinant PTZAB57R vector was constructed by the insertion of dsz A and B genes into the PTZ57R vector. The clones were confirmed by restriction digestion analysis. The PTZAB57R was extracted by large-scale method and was sequenced [MWG DNA Biotech]. Comparison of sequences of dsz A and B genes from native Rhodococcus FMF bacterium with Rhodococcus sp. IGTS8 bacterium were shown complete identity between them, which show that desulfurization pathway is conserved in this bacterium

Isolation of Aspergillus Niger Glucose Oxidase gene and cloning of that prokaryotic host

Glucose oxidase [GO] is an enzyme, which use in food, chemical and personal care industries as well as glucose diagnostic kits. The aim of this study was PCR-mediated amplification of GO gene from Aspergillus niger genome and cloning of it in E. coli as a basis for production of recombinant GO in Iran. A. niger [2029] was cultured in media containing peptone, glucose and malt extract in 24'C for 48-72 h. Genomic DNA was extracted by sonication and freeze-thaw in liquid nitrogen in a lysis buffer containing EDTA and SDS. GO gene was amplified with designed primers under optimized PCR condition. The PCR product was cloned in pTZ57R plasmid using InsT/Aclone[TM] [Fermentas] and the constructed plasmid was transformed into E. coli DH5. Map of this plasmid was confirmed by restriction analysis and named pTZ57RGO. Our data showed the methods used in this study was adequate for culture and extraction of DNA from filamentous fungi such as A. niger. We showed amplified DNA fragment has expected size in agarose gel electrophoresis and also the cloned GO has a correct size after restriction analysis. The GO gene isolated successfully from A. niger via optimized PCR conditions and was cloned in prokaryotic host. This is the first report of isolation and cloning of GO gene in Iran that can be used for further cloning of that gene in expression vectors for production of recombinant enzyme