NEW CELLULAR & MOLECULAR BIOTECHNOLOGY JOURNAL
Year:2013 | Volume:3 | Issue:9|Papers Count:13

Bacterial flagellum is a rotary nanomachin with a diameter of 45 nm that is located in cellular membrane and is used for movement in adjacent environment. The development of the first cellular motility apparatus has been accomplished during the first billion years of evolution as evidence by the high complexity of its structure for adaptation with environment. Unlike eukaryotic flagella that use a whip-like action to move whole cell, movement of a bacterial cell is generated by rotation of the filament. In the past researches, most of the flagella components and their functions are specified, whereas mechanisms of torque generation and rotation are unclear. A special apparatus assumes translocation and assembly of flagella components, which has homology with type III secretion system. For bacterial movement, the motors are energized by the transmembrane potential of specific ions, most commonly the proton motive force or the sodium motive force. The objective .of this paper is to discuss the recent finding by modem techniques during recent decades.

Aim and background: LEA proteins in wheat and cotton were identified and discussed as the first report in late embryonic proteins. Public classification for more LEA genes was inferred from the structure of the protein domain or chemically derived characters. Bioinformatics methods in genome research methods are useful in situations outside the laboratory.Material and Methods: In this study, LEA proteins for Wdhn13 genes in 8 wheat and wild wheat (including Triticum aestivum cv. Sardari, aestivum gonbad, durum Shosh, durum borojerd, urartu, dicocoides, tauschii and speltoides) were used and sequences from Wdhn13 was compared with Single gene sequences ofWdhn13 in NCBI.Results: The result of analysis showed that the Wdhn13 gene sequences in Sardari wheat were the most similar to the sequences in NCBI and the Wdhn13 gene sequences in urartu have the lowest similarity to the sequences in NCBI one. This difference is due to amino acid changes in the DNA sequence of samples. BLAST and Phylogenetic tree drawn from DNA sequence based on the UPGMA algorithm also confirmed this result. The Phylogenetic tree drawn with the logical evolution of the gene for the derivation of diploid species, and hexaploid, tetraploid was indicated. The table of sequence also confirmed that the sequence of the BLAST results and Phylogenetic trees are consistent over the time.Conclusion: The main result of this study was that given the amplification of target gene on the genomes of diploid, tetraploid and hexaploid species including genome AA and non-amplification on Species speltoides (BB) and tauschii (DD), it can be concluded that there is a target gene on the AA wheat genome.

Aim and background: The extra domain of the M2 protein of influenza is a weak immunogen, Hence this is not a suitable candidate for the vaccine development. The B subunit of the Cholera enterotoxin is nontoxic causing attachment of the holotoxin to the OMI at the surface of the epithelial intestine cells, while, CTB is highly immunogenic. So fusion of these two genes would be very applicable in influenza vaccine development.Material and methods: using PCR with specific primers including restriction sites ctxB was amplified. Standard strain of influenza type A (AJPUERTO RICO/8/34) was prepared and M2e was amplified using RT-PCR with specific primers. The second PCR reaction was performed for the fusion of the M2e and ctxB using the F primer of M2e and R primer of ctxB. PCR product was digested with BamHI and EcoRI and cloned into the pET28a. Verification of the cloning was performed using sequence analyzing. After analysis, the recombinant pET28a1M2e-ctxB was transferred into E. coli Top10. The final confirmation was performed using colony PCR, double digesting and sequence analysis.Results: Sequence analysis showed that M2e has been fused to ctxB in an exact frame. Moreover the fused gene was cloned into the restriction site of the BamHI and EcoR1 in pET28a.Conclusion: Despite stability of the sequence of M2e it is not immunogen but fusion to strong immunogens such as CTB would be a new suggestion for Influenza recombinant vaccine production.

Aims and Background: Mycoplasma contamination in cell culture is considered as a serious problem in the manufacturing of biological products. Our goal in this research is to find the best standard and a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in cell cultures of National Cell Bank of Iran. Materials and Methods: In this study, 40 cell lines suspected of mycoplasma contamination were evaluated by three different methods (microbial cultivation, enzymatic and molecular). Enzymatic evaluation was performed using Mycoalert® kit and in molecular technique, a universal primer designed based on common and fixed 16SrRNA ribosomal sequences was used. Results: Mycoplasma contamination in cell cultures with molecular, enzymatic and microbial cultivation methods were analyzed as 57.5%, 52.5% and 40% respectively. These results confirmed the higher rate of sensitivity, specificity and accuracy for molecular method in comparison with enzymatic and microbial methods. Conclusion: PCR assay based on fixed and common sequences in the 16SrRNA is a useful, valuable and reliable technique with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in cell cultures and other biological products. The enzymatic Mycoalert® method with respect to its sensitivity, specificity and very high speed detection of mycoplasma contamination (less than 20 minutes) after PCR can be used instead of methods. conventional microbial culture and DNA staining fluorochrome.

Aim and Background: Human serum albumin (HSA) is the most important components of the circulatory system. HSA can transport many metabolites and hormones in the body. It is effective for blood volume stabilization and the bum treatment. The preparation of HSA from human plasma is costly and the risk of contamination with various pathogens is high. Therefore, biotechnology could promise a new way to solve the problems.Materials and Methods: HSA gene was synthesized based on the sequence of the gene in NeBI website. HSA was cloned into pET22b expression vector and then the recombinant plasmid was transformed into BL2l (DE3) competent cell. The colony (containing recombinant expression vector) was cultured overnight. The culture was induced with IPTG 1.5 mM in the logarithmic phase (OD600=0.6). Sampling was carried out in different times and it was analyzed on %10 (w/v) SDS- PAGE. The expression of recombinant human serum albumin (rHSA) was confirmed by western blot. Finally, rHSA was purified by affinity chromatography.Results: In this study, human serum albumin was produced in the bacterial periplasmic environment. The highest expression level of rHSA was observed 24 h after the bacterial induction. Conclusion: In this study, rHSA was produced in E. coli host.

Aim and Background: Lipases are the valuable biocatalysts which have many applications in the industry. It is widely used in catalyzing chemical reactions such as water, alcoholic, acidic hydrolysis and also esterification. Bacterial lipases are the most important kinds of enzymes in the industry because they have special advantages such as the high stability in organic solvent, the ability of soft catalyzation of hydrolytic reactions, facility in production process and it's relatively low cost. Bacillus pumilus is one of the most considerable bacterial sources of this enzyme but the main problem for producing this enzyme is low expression and secretion. The aim of this research is the production of recombinant lipase from native strain in secreting manner for increasing the level of expression in Bacillus subtilis.Materials and Methods: Lipase-coding gene of bacillus pumilus was cloned into pWB980 plasmid by genetic engineering. After this step, construction plasmids were transferred to Bacillus subtilis WB600 as a host. Then expression and secretion level between native and recombinant forms was evaluated and compared.Results: Secretory form of lipase enzyme was expressed in Bacillus subtilis. An expression level of this enzyme is higher than normal strain.Conclusion: Recombinant lipase production in Bacillus subtilis is a suitable method for increasing the expression of this enzyme.

Aim and Background: Phytic acid is an important mineral compound which can be preserved in plants. Phytase (E.C. 3.1.3.26 or E.C. 3.1.3.8) has been used as a cereal feed additive that can enhance the phosphorus and mineral absorption in monogastric animals food; so it can reduce the phosphorus output into the environment. In this research, Cloning of gene encoding phytase containing hexahistidine-tag was conducted in Escherichia coli and then yeast for purification of the produced enzyme after expression in this yeast.Material and method: Sequence of protein encoding Peniophora lycii fungal phytase, was obtained from protein data bank, designed and then synthesized according to the expression codon usage of the yeast. A piece of DNA containing hexahistidine-tag was added into the pFPMTMF aa shuttle vector fallowed by transformation through electroporation method into the expression yeast.Result: Recombinant DNA encoding phytase containing hexahistidine-tag was produced. Presence of gene in bacteria and yeast was proved with double digest and PCR methods. Conclusion: Successful cloning of fungal phytase gene containing hexahistidine-tag was conducted in Escherichia coli bacteria and then yeast for high level expression and purification of this enzyme.

Aim and Background: Proteases have multiple applications within organic media, and then their organic solvent stability is critically important. Pseudomonas aeruginosa elastase is an organic solvent stable enzyme. In the present work, following protein purification, its organic solvent activity and stability have been investigated. Materials and Methods: Protein purification was carried out by affinity chromatography. Enzyme activity within organic solvent media has been measured by activity measurements in the 0-70% (VN) of organic solvents including ethanol, methanol, isopropanol, dimethyl formamide (DMF), glycerol and ethylene glycol. Results: In the presence of all concentrations of organic solvents investigated in the present study, except ethanol, activity of elastase was very high and even higher than that of control, i.e. in the absence of organic solvents. For instance, in the presence of 70% (VN) of methanol, DMF and glycerol enzymatic activity was 3.5, 1.4 and 1.2 times higher than that of 0% of organic solvents. Conclusion: On the contrary to most enzymes whose activity diminishes in organic solvents, the present study clearly indicated that elastase activity increases even in the presence of organic solvents. This finding means that Pseudomonas aeruginosa elastase is not only an organic solvent enzyme, but more ideal catalyst within aqueous-organic than absolutely aqueous medium.

Aim and Background: Biosurfactants are a group of secondary metabolites with surface active properties like reducing superficial and interfacial tensions that are synthesized by a variety of micro-organisms. Interest in their potential applications by various industries has significantly increased recently, particularly because of their advantages such as lower toxicity, biodegradability and effectiveness at a wide range of pH and temperature values. Use of expensive substrates is among several economic limitations for large scale production of biosurfactants. A suggested strategy to solve this cost problem is the use of inexpensive substrates such as lipidic wastes. This research aimed to evaluate the vegetable oil refinery wastes as inducing substrates for rhamnolipid biosynthesis by Pseudomonas aeruginosa MR01, and their transformation to high value-added materials.Materials and Methods: In this research, fatty acids present in vegetable oil refinery wastes and total organic materials were measured using Gas-Chromatography and COD, respectively. Biosurfactant emulsification was determined through the emulsification index (E24). Results: COD for oil wastes showed high values around 2,000,000 and emulsification was obtained on average about 40%.Conclusion: A dramatic emulsification activity of rhamnolipid biosurfactants produced by high COD oil wastes represented a promising green material to reduce the problems regarding the wastes and hydrophobic pollutants accumulation.

Aim and Background: Recently, application of SO2 during cold storage is restricted since its residues are toxic and dangerous to human health. Salicylic acid (SA) is an alternative method to SO2 and can alleviate post harvest oxidative stress. Our research goal, in the present study, was to characterize the effects of salicylic acid (SA) on some anti oxidative characteristics of grape (Vitis vinifera L. cv Bidaneh Sefid, Dastrchyn, Sahebi and Qizil usum) fruits applied with or without SA under cold storage for two weeks.Materials and Methods: The selected clusters were randomly divided into two groups so that SA-treated group immersed for ten minutes in solutions of 1 mM SA and were air-dried at room temperature.Results: Salicylic acid increased the relevant water content (RWC) of Sahebi and Qizil usum in comparison to those of control at the end of the shelf life, whereas the effect of SA treatment on relative water content of the other cultivars was not significant during storage life. In Bidaneh Sefid, no difference in superoxide dismutase (SOD) activities between SA-pretreated and control fruit was observed, whereas SOD activity in SA-pretreated Dastrchyn was significantly higher than that in the controls during 15 days of postharvest life.Conclusion: In this work, SA considerably reduced the rise in malondialdehyde (MDA) content in cultivars Sahebi and Qizil usum during storage, obviously because of an efficient scavenging following significant enhancement of SOD and catalase (CAT) activity. These results suggest that SA application can improve antioxidant defense system of Sahebi and Qizil ilsum during storage, and it may be recommended for increasing storage life and maintaining their quality.

Aim and Background: In the past decade, science and technology has put enormous attention to produce nanoparticles. There are many methods to synthesize nanoparticles. Many techniques are inefficient in terms of material and energy consumption. Most chemical methods are hazardous chemicals for humans and the environment. For these reasons, there is a demand to produce nanoparticles with biological methods. This research is concentrated on extracellular biosynthesis of silver nanoparticles using non-pathogenic species of Leishmania.Materials and Methods: It was found that aqueous silver ions are reduced in solution when exposed to protozoa Leishmania, thereby leading to the formation of silver nanoparticles. The formation of silver nanoparticles was analyzed by uv-vis spectroscopy, microscopic images and elemental X-ray scattering spectra.Results: In UV-Vis spectroscopy, the peak at 420 nm corresponds to the surface plasmon resonance of silver nanoparticles. The Atomic Force microscope (AFM) and Scanning electron microscope (SEM) images of nanoparticles in aqueous solution showed the production of silver nanoparticles (average particle size: 2080- nm) by the protozoa. EDX spectra of silver nanoparticles excited by an electron beam, showed peaks for the elements of Ag.Conclusion: Leishmania protozoon is a green factory for the production of silver nanoparticles. Silver nanoparticles were in the range of 20-80 nm in dimensions. The use of this protozoon for silver nanoparticles synthesis offers the benefits of eco-friendliness, amenability, and timesaving for large- scale production.

Aim and Background: Menstrual disorders such as premenstrual syndrome (PMS) is common in adolescents and young women. Premenstrual syndrome and premen strualdy sphonic disorder are series of mood swings, behavioral and physical changes that have a negative impact one motions and performance in women. The purpose of this study was to estimate the signs, symptoms, prevalence, frequency and severity of PMS and its effects on the life quality.Materials and Methods: 116 students from the Islamic Azad University of Parand were chosen randomly and asked to fill up the questionnaire. The data were analyzed by SPSS (Version 16) using logistic regression. According to the results, 84.2% of the girls had disorders of PMS and out of this 62.5% had PMS, whereas 37.5% had PMDD. Also, 80.7% of them were single and 19.3% were married which 84.78% of singles and 81.81% of married women had PMS.Results: There was no positive association between age, marriage status and the period of menstrual with this syndrome (P>0.05). The most common symptoms included snervousness/ irritability, abdominal and back pain, fatigue/decreasedenergy, decreased interest in working relationships, stress and anxiety, jointormusclepain, tenderness and breast pain.Conclusion: Symptoms like nervousness/irritability, abdominal and back pain were observed among women and according to these data we came to know that physicians should pay attention not only to check for the physical signs but also for moral and behavioral signs of people with PMS.