Abstract: In order to ensure the biosafty of the IFN-γ antiviral activity assay, we used a replication-deficient VSV carrying GFP as an interferon sensitive indicator virus (VSV△G*G). The antiviral activities of porcine IFN-γ expressed in Escherichia coli and in baculovirus on MDBK cells were assessed. The results showed that the antiviral activity of porcine IFN-γ expressed in baculovirus could reach 105 IU/mL, while the porcine IFN-γ expressed in E. coli showed some antiviral activity (32 IU/mL) after refolding. The results of the VSV△G*G-based antiviral assay were almost identical to that of the VSV*GFP-based assay, suggesting it is highly feasible to use VSV△G*G as a substitute for VSV*GFP, making assays for IFN-γ antiviral activity safer and more accurate.
Abstract: Most studies related to determining the expression profile of genes and specific promoters used histochemical localization of the reporter gene, gusA. While the histochemical method for visualizing gusA expression suffers from several limitations in the determination of gene expression and location, especially in the tissues with high background acitivty. To solve this problem, a transient expession vector pBI221-RFP/GFP, was constructed using GFP and RFP as double fluorescent marker genes. This vector used CaMV 35S promoter to drive GFP and determine the transforming efficiency. It analyzed expression profile of the target gene and promoter through the RFP activities of the tranformed tissues. Through the specific promoter AGPL1 from watermelon and E8 promoter from tomato, it is resistible to use this vector to study the expression patterns of promoters. Results indicated that the pBI221-RFP/GFP is a very efficient transient expression vector that can be verify the functions of the genes and promoters.
Abstract: Resveratrol is a naturally occurring stilbene, a kind of polyphenolic compounds, found in a limited number of plant species such as grape, peanut, and pine. It has been considered as a phytoalexin in plants, and many studies have also shown its health benefits such as antioxidant activities, cancer prevention, blood thinning, and life span extension. This paper reviews the characteristics of resveratrol in aspects of synthesis, extraction, purification, and determination. In particular, the new outcomes of physiology function and the transgenic approaches have been presented. The challenges and chances for genetic engineering and heath-related industries were also discussed.
Abstract: We found seven tag sequence with high homology in dbEST by using human musclin gene, and got its cDNA sequence, which consists of 651bp and the open reading frame was 54~452 bp detected by RT-PCR, encoding 132 amino acid residue protein . The new gene has high homology with that of human, mouse and rat, the rate is 87.2%, 77.6% and 77.9%, respectively; the gene fragment was cloned into expression vector pGEX-4T-1, and the recombinant was transformed into E. coli BL21. Induced by IPTG, the fusion protein GST-musclin, a 38.59 kD protein was successfully expressed in E. coli BL21 and identified by Western blotting.
Abstract: BMP6 is a potent protein for future treatment strategies of bone regeneration as it is a very important regulator of bone homeostasis. Active BMP6 is a dimer containing multidisulfide bonds and is a highly hydrophobic protein prone to aggregation. To obtain soluble and active BMP6 in Escherichia coli, we compared the effects of four N-terminal fusion tags (TRX, GST, MBP and CBD) and N-terminal His6-tag. The expression and solubility were tested under the different conditions (expression hosts, temperatures and inductor concentrations). A series of experiments leads to the finding that the placement of MBP before the BMP6 is best in availing the soluble expression of the protein. Our study alsodemonstrates that in E. coli BL21trxB(DE3) cytoplasm, which is a thioredoxin reductase mutant strain, soluble homodimeric BMP6 can be formed. The overexpressed MBP-BMP6 fusion protein is purified by chromatography, and shown to be functionally active.
Abstract: The somatic cells can be induced into ES-like stem cells when retrovirally infected the defined transcription factors including Oct4, Sox2, Klf4 and c-Myc. These ES-like cells are named induced pluripotent stem (iPS) cells and this method is called iPS technology. Until the end of 2009, iPS cell lines have been generated in various animal species, such as mouse, human, rhesus monkey, rat and pig. Mouse iPS cells are also used to generate chimera mice and viable mice through the tetraploid complementation. Although iPS cells are extremely similar to ES cells in both morphology and growth features, to generate iPS cells do need the defined culture procedures. Based on the update global iPS technology development and the iPS studies in our laboratory, this paper focused on the establishment of iPS cell lines and improvement of iPS cell culture condition.
Abstract: To investigate the effect of Kozak sequence (+4A or +4G) on expression of green fluorescent protein (GFP) gene in HEK293 cells. The eukaryotic expression vectors containing GFP gene with different Kozak sequence (+4A or +4G) were constructed by classic DNA recombination methods, including PCR, enzyme digestion, ligation, transformation, identification, et al. Two different Kozak sequences (+4A or +4G) were obtained through PCR with different mutagenic primers. The right recombinant plasmids pHGFP-A and pHGFP-G were transfected into HEK293 cells by liposome-mediated gene transfer method. The expression level of GFP was observed by fluorescent microscope, flow cytometry and Western blot. The flow cytometry revealed that the expression levels of GFP fluorescence in pHGFP-A and pHGFP-G transfected cells were about 15% and 45%, respectively. Western blot showed the specific bands of about 27 kD (GFP) both in pHGFP-G and pHGFP-A sample lanes; and the GFP expression density of pHGFP-G was about 3.87-fold as that of pHGFP-A by ImageJ software analysis. These results indicated that the +4G in Kozak sequence (when ?3 site is purine base pair) plays an important role in GFP protein translation, which enhances the GFP expression up to 4-fold in HEK293 cells.
Abstract: Laccases (benzenediol: oxygen oxidoreductases; EC 1.10.3.2) are copper-containing polyphenol oxidases that can oxidize a wide range of aromatic compounds, concomitantly with the transfer of four electrons and the reduction of molecular oxygen to water. The progress on the research of laccases structure and function is reviewed. Their three-dimensional structures and catalytic mechanism, as well as their applications in different fields are emphasized.
Abstract: Microbe is extremely abundant in nature, and its size has a very wide coverage from nano- to micro-scale making it suitable to be processed at multi-scale level as natural "building blocks" and "chassis cells". Biofabrication based on microbes is an artificial manipulation on microbes to assemble functional materials and devices by using the specific structures and various biological functions of microbes. In the meantime, the novel strategies of biofarication enables us to study the behavioral details of microbes, which will provide new platforms for uncovering the unsolved basic scientific problems of microbes. In this paper, we reviewed the frontier and progress in biofabrication from nano- and micro-scale in microbes that were manipulated as structured "building blocks" or functional "micro/nano robots".
Abstract: Bacterial promoter is a kind of regulators which are needed in bacterial gene expression and decide the strength and opportunity of gene expression. By insertion or deletion of promoters, we can change bacterial gene expression in order to study the growth and metabolic regulation. Promoters are also used to construct many kinds of vectors, so as to express heterologous genes. The study of promoter recognition and application is of great importance to realize the regulation of genes, gain products effectively and promote biological catalysis and metabolic engineering. This paper reviews bacterial promoters, and the methods for recognition of bacterial promoters as well as the study and application of bacterial promoters.
Abstract: For the first time ever, the scientists of J. Craig Venter team have created actual self-replicating synthetic life. The research was just published in the Journal of Science on May 20, 2010. Although this news immediately brings the worry about the possible potential threat to biosecurity and biosafety as well as the ethical disputes, it yet indicates that mankind have made a new step forward in synthetic biology. In the time of post-genome era, we believe the advancement of synthetic biology that might affect or change the future life of human being will be widely used in energy, environment, materials, medication and many other fields.
Abstract: Gene expression is regulated by different transcriptional regulators. The transcriptional regulator isocitrate lyase regulator (IclR) of Escherichia coli represses the expression of the aceBAK operon that codes for the glyoxylate pathway enzymes. In this study, physiological and metabolic responses of the deletion of the iclR gene in E. coli BW25113 were investigated based on the quantification and analysis of intracellular metabolic fluxes. The knockout of the iclR gene resulted in a decrease in the growth rate, glucose uptake rate and the acetate secretion rate, but a slight increase in biomass yield. The latter could be attributed to the lowered metabolic fluxes through several CO2 generating pathways, including the redirection of 33% of isocitrate directly to succinate and malate without CO2 production as well as the reduced flux through the pentose phosphate pathway. Furthermore, although the glyoxylate shunt was activated in the iclR mutant, the flux through phosphoenolpyruvate (PEP) carboxykinase kept almost unchanged, implying an inactive PEP-glyoxylate cycle and no extra loss of carbon atoms in the mutant strain. Both the reduced glucose uptake rate and the active glyoxylate shunt were responsible for the minor decrease in acetate secretion in the iclR knockout strain compared to that in the wild-type E. coli strain.
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