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Results for “strain engineering”

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At least 343 records · Page 19Linked to original sources

PubMed2004

[Effectiveness for medicine wastewater treatment biotechnology].

The effectiveness for the XZEH medicine wastewater treatment biotechnology was conducted. The results show that it needs to increase 28% of the aeration tank volume in order to meet the effluent COD concentration at the first grade wastewater discharge standard level in China. If the engineered strain Xhhh and informatics software (Ebis) were used, the specific degradation rate of Xhhh strain may be higher 128% than that of the natural bacterium XZ strain and the aeration tank needed may be 51% decreased. This should be a potential technical patch for improving the biotechnological effectiveness and making the effluent at the first grade discharge standard.

Biodegradation, Environmental↗

PubMed2005

Cloning of delta6-desaturase from Mucor circinelloides and its high expression in Saccharomyces cerevisiae.

The aim of this study is to obtain Saccharomyces cerevisiae engineering strain with high gamma-linolenic acid (GLA, gamma-C18:3), which is a nutritionally important fatty acid that plays a vital role in biological structure and cell functions. As the first step,we cloned gamma6-desaturase gene from fungus mucor circinelloides by RT-PCR; delta6-desaturase is responsible for the transformation of linoleic acid into GLA. The PCR product was subcloned into yeast expression vector pYES2 to generate a recombinant plasmid pYES412. Transformation of S. cerevisiae strain INVSc1 was done by the lithium acetate method and the recombinant yeast cells were selected on a uracil-deficient medium. On appropriate medium and temperature,linoleic acid was provided as a substrate to yeast cultures,and the level of gamma-linolenic acid reached 50.07%. So far,the result we obtained is the best in terms of the level of expression of delta6-desaturase gene in Saccharomyces cerevisiae.

Cloning, Molecular↗

PubMed2005

Molecular genetic advances in sleep research and their relevance to sleep medicine.

Neurobiology has been profoundly impacted by the availability of molecular genetic techniques within the past decade, and the sleep research field is no exception to this trend. Genetic influences on normal and pathologic sleep have long been recognized, but only recently have the contributions of discrete genetic loci to the regulation of sleep timing and sleep-related physiologic phenomena begun to be demonstrated, particularly through the use of genetically engineered strains of rodents. The ability to monitor the expression of a large number of genes has also accelerated in recent years, leading to a better understanding of the molecular and neurochemical correlates of sleep and of sleep loss. This article reviews the contributions of molecular genetic studies to our understanding of the processes that underlie sleep and its pathologies, discusses their relevance to the treatment of sleep disorders, and offers some speculation on how the sleep field might further be advanced through molecular genetic analysis.

Animals↗

PubMed2005

[Cloning and characterization of an harpin-encoding gene from Xanthomonas axonopodis pv. glycines required for hypersensitive response on nonhost plant tobacco].

An hpa1 gene was cloned into an expression vector, pET30a(+), from the genomic DNA of Xanthomonas axonopodis pv. glycines (Xag), the causal agent of soybean bacterial pustule, with degenerated primers by polymerase amplification reaction (PCR). The gene product was extracted from the conjugate (BHR-3) of BL21 (DES) with the recombined vector pHR3 after the engineering strain was induced by IPTG in LB medium. The SDS-PAGE gel showed that the gene product was 15.1kD. The product was heat-stable (10 min at 100 degrees C), protease K sensitive, and able to trigger hypersensitive response (HR) in common tobacco, but was unable to elicit HR in NahG transgenic tobacco in which salicylic acid accumulation was abolished. Moreover, the HR elicitation of the protein in tobacco was dispelled by eukayotic metabolic inhibitors, actinomycin D, cycloheximide and LaCl3. The 402 bp hpa1 gene in this study putatively encoded a 133 ammonia acid protein of which glycine (G) was rich with 21.1%. Sequence comparison indicated that the hpa1 gene and its protein was 51.4% - 93.8% identity with those of Xanthomonas oryzae pv. oryzae and other Xanthomonas species and pathovars. Alignments of harpin proteins of Xanthomonas genus displayed that the glycine-rich region with GGG-GG motif was variable. The comparison also showed that the harpin-encoding gene of Xag (nominated here as hpa1(Xag)) did not possess any similarity with that of Erwinia amylovora, Pseudomonas syringae and Ralstonia solanacearum at nucleotide and protein levels. It is concluded that hpa1(Xag) gene encodes an harpin protein which elicits a typical HR in nonhost tobacco.

Amino Acid Sequence↗

PubMed2005

[Construction of an engineered Acidithiobacillus caldus with high-efficiency arsenic resistance].

Using the recombinant technique in vitro, a new arsenic resistance plasmid pSDRA4 was constructed by subcloning the arsenic resistance genes from plasmid pUM3 into the wide-host-range IncQ plasmid pMMB24 with the hybrid trp-lac ( tac ) promoter, and followed by deleting the regulative gene of the promoter, the lacIQ gene. Then plasmid pSDRA4 was introduced from E. coli into extremely acidophilic obligately chemolithotrophic Acidithiobacillus caldus by conjugative transfer with a frequency of( 1.444 +/- 0.797) x 10(-4), and the engineered strain of Acidithiobacillus caldus (pSDRA4) for biomining was constructed. The successful transfer demonstrates the development of a conjugational system between strains of E. coli and A. caldus. The recombinant plasmid pSDRA4 is stable in A. caldus. Compared with wild type A. caldus, the level of the arsenic resistance of A. caldus (pSDRA4) is greatly raised from 10mmol/L to 45mmol/L.

Acidithiobacillus↗

PubMed1990

Efficacy of a killed gpX deleted pseudorabies virus vaccine.

Ten inactivated vaccines containing one of four adjuvants and varying concentrations of pseudorabies virus (PRV) antigens were compared in order to select a vaccine suitable for commercial production. A genetically engineered strain of PRV lacking the gene coding for glycoprotein X (gpX) was used in these vaccines. Vaccinated pigs were challenged intranasally with virulent PRV to determine the efficacy of vaccines. Vaccination of pigs with one dose of experimental vaccines adjuvanted with 50% Montanide ISA 50 or 20% Syntrogen induced a protective immunity at least equal to that induced by two commercially available killed PRV vaccines also evaluated. An experimental vaccine containing 20% Syntrogen was selected and further evaluated according to United States Department of Agriculture licensing requirements. None of the pigs vaccinated with this vaccine produced gpX antibodies detectable by the HerdChek: Anti-PRV-gpX assay. Therefore, this assay could differentiate PRV vaccine induced antibodies from antibodies induced by natural exposure when used in conjunction with this killed gpX deleted PRV vaccine.

Animals↗

PubMed1987

Mechanism of Escherichia coli alpha-hemolysin-induced injury to isolated renal tubular cells.

Alpha-hemolysin (AH) is a 110,000-dalton protein secreted extracellularly by certain Escherichia coli. This protein is an acknowledged virulence factor for E coli and recently has been implicated as an important determinant in the pathogenesis of E coli pyelonephritis. Recombinant engineered strains of E coli were used that varied only in their ability to secrete AH extracellularly. The effect of AH on vital dye exclusion, oxygen consumption rate (QO2) adenosine triphosphate (ATP) levels, superoxide (O2-) and hydrogen peroxide (H2O2) production in preparations of isolated rat cortical renal tubular cells (RTCs) was assessed. Approximately 5-10 pg of AH dramatically stimulated QO2 by nearly 150%. This was associated with a marked increase in production of O2- and H2O2, to 13.9 +/- 1.7 and 13.2 +/- 2.1 nM/mg cell protein, respectively (P less than 0.05), as well as a 38% decrease in cellular ATP. These biochemical effects were all seen after a 30-minute exposure to AH and by 120 minutes were associated with 15.7% +/- 1.1% of RTCs that were unable to exclude vital dye. The effect of AH on QO2 and O2- formation was prevented by pretreatment of RTCs with ouabain, which indicates that the effect of AH on oxygen metabolism is linked to Na-K ATPase activity. However, when ouabain-treated RTCs were exposed to AH, ATP remained depressed despite the inhibition of QO2 and O2- production. In contrast, in ouabain-pretreated RTCs, cell membrane integrity was dramatically protected, because only 2.4% +/- 0.4% of RTCs were not unable to exclude vital dye. Thus, the data demonstrate that E coli AH provokes at least two biochemical events that may be injurious to RTC: increased oxygen intermediates (O2- and H2O2 and ATP depletion. These findings with ouabain suggest that the first mechanism of injury may be a more proximate cause of cell death. Moreover, the data suggest that endogenous production of reactive oxygen molecules may be critical modulators of RTC membrane injury.

Adenosine Triphosphate↗

PubMed1995

Molecular cloning and expression of human granulocyte-macrophage colony stimulating factor in Escherichia coli system.

Human granulocyte-macrophage stimulating factor (hGM-CSF) is an important hematopoietic growth factor. hGM-CSF is able to stimulate hematopoietic progenitors to produce granulocytes, macrophages and eosinophils. hGM-CSF also shows the potential to treat myeloid leukemias. In order to prepare enough hGM-CSF, a genetically engineered strain E. coli HB101/pZW.GM47 was constructed by DNA recombination technologies in the present study, and characterized by restriction analysis, DNA sequencing, SDS-PAGE, Western blotting and bioassays. The strain has been shown to produce a 14kDa mature hGM-CSF protein with expression level higher than 40% and a specific activity of 5.0 x 10(7) u/mg which shows a good potential for clinical applications.

Bacterial Proteins↗

PubMed1994

[Role of EcoRI-BclI fragment at the promoter upstream region on the expression of alpha-amylase gene].

A series of plasmids derived from pNL201 with the promoter upstream region of alpha-amylase gene of B. licheniformis have been constructed. In the meantime, a series of plasmids derived from pAmy41 with full alpha-amylase gene have also been constructed and they have different promoter upstream region with the EcoRI-BclI fragment deleted or partial destroyed. The alpha-amylase activity of the engineering strains carrying alone or double plasmids has been determined. The statistical results show that the EcoRI-BclI fragment possesses negative regulation in the expression of alpha-amylase gene in B. subtilis.

Deoxyribonuclease EcoRI↗

PubMedDate not supplied

Cephalosporin acylases: enzyme production, structure and application in the production of 7-ACA.

Cephalosporin acylases have application in the production of 7-aminocephalosporanic acid which forms a key raw material for the preparation of semisynthetic injectable cephalosporins. The enzymes are of industrial importance and hyperproducing genetically engineered strains have been constructed. Different aspects of these enzymes such as subunit structure, post translational modification, primary structure, substrate specificity and their importance in pharmaceutical industry are discussed.

Amino Acid Sequence↗

PubMed1996

Expression of acylphosphatase in Saccharomyces cerevisiae enhances ethanol fermentation rate.

Previous experiments in vitro have demonstrated the ability of acylphosphatase to increase the rate of glucose fermentation in yeast. To evaluate the possibility of increasing fermentation in vivo also, a chemically synthesized DNA sequence coding for human muscle acylphosphatase was expressed at high level in Saccharomyces cerevisiae. Ethanol production was measured in these engineered strains in comparison with a control. Acylphosphatase expression strongly increased the rate of ethanol production both in aerobic and anaerobic culture. This finding may be potentially important for the development of more efficient industrial fermentation processes.

Acid Anhydride Hydrolases↗

PubMed1996

Cloning and expression of full-length delta-endotoxin cryIA(c) gene in three kinds of prokaryotic systems using shuttle vector pHT3101.

Two fragments, 6.5kb and 4.3kb encoding 5' end and 3' end of delta-endotoxin cryIA(c) gene, respectively, were selected from the Bacillus thuringiensis kurstaki-HD-73 75kb plasmid gene pool using random primer labelling delta-endotoxin cryIA(b) gene probe. The full-length 3.92kb cryIA(c) gene including 5' end 152 bp promoter sequence and 3' end 198 bp terminater sequence was rebuilt after uncoding sequences were deleted. Three kinds of engineering strains harbouring the same recombinant plasmid pHTY1 were obtained after the cryIA(c) gene had been subcloned in shuttle vector pHT3101 and transformed to E. coli NM522, Bacillus subtilis AS1176 and Bacillus thuringiensis crystal-deficient 4D10(H3ab). SDS-PAGE electrophoresis patterns reveal that the cryIA(c) gene expressed the same 133,300 protoxin proteins in all three host systems with the same molecular weight to the crystal protein from the Bacillus thuringiensis kurstaki HD-73. Immuno-assays indicate that the expression proteins can react with antiserum of HD-73 paraspore crystal protein in the same pattern as the natural crystalprotein. Bioassays using crude expressed products from three host strains reveal that they all showed toxicities to second instar larvae of Plutella xylostella, and their LD50 were calculated to be 0.311 micrograms/cm2, 0.02 micrograms/cm2 and 0.017 micrograms/cm2, respectively.

Bacillus subtilis↗

PubMed1996

The construction of a stable starch-fermenting yeast strain using genetic engineering and rare-mating.

To develop a yeast strain that is able to produce ethanol directly from starch, alpha-amylase cDNA (originated from mouse salivary glands) was introduced into the hyploid Saccharomyces diastiticus cells secreting glucoamylase by using a linearized integrating vector. The integrating vector contains a LEU2 gene and the inside of the LEU2 gene was cut by KpnI to make the linearized vector. One of the transformants exhibited 100% mitotic stability after 100 generations of cell multiplication. To improve its ethanol-fermentability, the haploid transformant was rare-mated with a polyploid industrial strain having no amylase activity. The resulting hybrid RH51 produced 7.5 (w/v) ethanol directly from 20% (w/v) soluble starch and its mitotic stability was 100% at the end of fermentation.

Animals↗

PubMed1992

Use of a genetically engineered Escherichia coli strain to produce 1,2-dihydroxy-4'-chlorobiphenyl.

Genetically engineered kanamycin-resistant Escherichia coli HB101 containing the mutant chimeric plasmid pAW6194-T17 specifying biphenyl dioxygenase and dihydrodiol dehydrogenase and lacking the ability to produce active 3-phenylcatechol dioxygenase was used to produce 1,2-dihydroxy-4'-chlorobiphenyl (DHCB) from 4-chlorobiphenyl. Resting-cell suspensions of genetically engineered E. coli in mineral salts medium (pH 7.0) containing 880 microM 4-chlorobiphenyl produced 110 microM DHCB. The Km for 4-chlorobiphenyl was 3.3 mM. Biotransformation of DHCB from 4-chlorobiphenyl was maximum when cells (2.5 mg of protein per ml) were incubated with shaking (150 rpm) at pH 7.0 and 30 degrees C for 6 h. The enzymatically produced DHCB was a suitable substrate for assaying 3-phenylcatechol dioxygenase activity. Biologically produced DHCB showed UV and mass spectra similar to those of chemically synthesized DHCB. The bioconversion rate of ortho-substituted chlorobiphenyl was slower than that of the para- or meta-substituted chlorobiphenyl.

Biotransformation↗

PubMedDate not supplied

Metabolically engineered Rhodobacter sphaeroides RV strains for improved biohydrogen photoproduction combined with disposal of food wastes.

Three differently metabolically engineered strains, 2 single PHA- and Hup- mutants and one double PHA-/Hup- mutant, of the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides RV, were constructed to improve a light-driven biohydrogen production process combined with the disposal of solid food wastes. These phenotypes were designed to abolish, singly or in combination, the competition of H2 photoproduction with polyhydroxyalkanoate (PHA) accumulation by inactivating PHA synthase activity, and with H2 recycling by abolishing the uptake hydrogenase enzyme. The performance of these mutants was compared with that of the wild-type strain in laboratory tests carried out in continuously fed photobioreactors using as substrates both synthetic media containing lactic acid and media from the acidogenic fermentation of actual fruit and vegetable wastes, containing mainly lactic acid, smaller amounts of acetic acia, and traces of higher volatile acids. With the lactic acid-based synthetic medium, the single Hup- and the double PHA-/Hup- mutants, but not the single PHA- mutant, exhibited increased rates of H2 photoproduction, about one third higher than that of the wild-type strain. With the food-waste-derived growth medium, only the single Hup- mutant showed higher rates of H2 production, but all 3 mutants sustained a longer-term H2 photoproduction phase than the wild-type strain, with the double mutant exhibiting overall the largest amount of H2 evolved. This work demonstrates the feasibility of single and multiple gene engineering of microorganisms to redirect their metabolism for improving H2 photoproduction using actual waste-derived substrates.

Acyltransferases↗

PubMed2005

Engineered Saccharomyces cerevisiae strain BioS-1, for the detection of water-borne toxic metal contaminants.

Saccharomyces cerevisiae responds to extracellular toxic stimuli by increasing intracellular cyclic AMP levels, leading to activation of a cAMP-dependent protein kinase, protein kinase A (PKA). Activated PKA phosphorylates downstream substrates, including specific DNA-binding proteins, to turn off the expression of most or all of the yeast genes. Such cAMP-PKA-mediated inhibition of gene expression in response to toxic stimuli appears to be unique to S. cerevisiae. For instance, in mammalian cells, the cAMP-PKA signaling pathway is rather responsive to growth factors and hormones in addition to being primarily involved in the activation of gene expression. Activation of gene expression by the cAMP-PKA pathway in mammalian cells is due mainly to the presence of cAMP-response elements (CREs) located in the promoters of many mammalian genes, and the expression of PKA-responsive stimulatory transcription factor CRE-binding protein, commonly referred as CREBP, which binds to the CREs. Thus, activation of the cAMP-PKA signaling pathway results in the phosphorylation of CREBP by PKA, and phosphorylated CREBP transactivates specific gene expression by interacting with the cognate CRE. Based on these findings, we sought to engineer a yeast-based biosensor, in which the stress-sensing cAMP-PKA pathway of yeast is coupled to the mammalian CREBP-CRE-stimulated gene expression pathway, which drives the expression of a reporter protein, such as green fluorescent protein (GFP). As a primary step toward the development of this biosensor, we engineered a yeast strain, BioS-1, by genetically altering YPH 501, a wild-type strain of S. cerevisiae, to express human CREBP and human CRE promoter-driven GFP. Exposure of BioS-1 to varying concentrations of As3+, Fe2+, Pb2+, and Cd2+ elicits concentration-dependent expression of the GFP reporter that can be easily monitored by the fluorescence emitted by GFP. The results also indicate that the engineered BioS-1 yeast cells can detect 2.5 ppm of these toxic metals and report it through the expression of GFP within 3 h. The results presented herein demonstrate that this engineered yeast strain can detect metal toxicants and can validate the use of this prototypic yeast strain to develop a biosensor that can be used to detect and monitor cytotoxic water-borne toxic heavy metals.

Biological Assay↗

PubMed2004

Biosynthesis of poly(3-hydroxybutyrate- co-3-hydroxyalkanoates) by metabolically engineered Escherichia coli strains.

Biosynthesis of polyhydroxyalkanoates (PHAs) consisting of 3-hydroxy-alkanoates (3HAs) of 4 to 10 carbon atoms was examined in metabolically engineered Escherichia coli strains. When the fadA and/or fadB mutant E. coli strains harboring the plasmid containing the Pseudomonas sp. 61-3 phaC2 gene and the Ralstonia eutropha phaAB genes were cultured in Luria-Bertani (LB) medium supplemented with 2 g/L of sodium decanoate, all the recombinant E. coli strains synthesized PHAs consisting of C4, C6, C8, and C10 monomer units. The monomer composition of PHA was dependent on the E. coli strain used. When the fadA mutant E. coli was employed, PHA containing up to 63 mol% of 3-hydroyhexanoate was produced. In fadB and fadAB mutant E. coli strains, 3-hydroxybutyrate (3HB) was efficiently incorporated into PHA up to 86 mol%. Cultivation of recombinant fadA and/or fadB mutant E. coli strains in LB medium containing 10 g/L of sodium gluconate and 2 g/L of sodium decanoate resulted in the production of PHA copolymer containing a very high fraction of 3HB up to 95 mol%. Since the material properties of PHA copolymer consisting of a large fraction of 3HB and a small fraction of medium-chain-length 3HA are similar to those of low-density polyethylene, recombinant E. coli strains constructed in this study should be useful for the production of PHAs suitable for various commercial applications.

3-Hydroxybutyric Acid↗

PubMed2005

A stimulation unit for the application of mechanical strain on tissue engineered anulus fibrosus cells: a new system to induce extracellular matrix synthesis by anulus fibrosus cells dependent on cyclic mechanical strain.

A bioreactor system consisting of a multifunctional stimulation unit and common 6-well culture plate is introduced to activate extracellular matrix synthesis in intervertebral disc cells due to cyclic mechanical strain. The developed stimulation unit is sterilizable and reusable. It is viable for cultivation and mechanical stimulation of cartilage tissue and tissue engineered cell matrix constructs in combination with the common 6-well culture plate. The custom made device allows long-term cultivations in batch- or continuous operation mode. Manual handling and thereby the risk of contamination is reduced. Sampling, changing the medium, and addition of supplements are easily performed from the connected conditioning vessel. This bioreactor system enables stimulation of different samples independently during one run. For the work presented here anulus fibrosus cells from pigs were taken and immobilized in agarose to obtain three-dimensional cell matrix constructs. Over a period of 14 days the constructs were subjected to 10% compression under cyclic mechanical pressure with a frequency of 0.1 Hz. Afterwards the constructs were biochemically examined for hydroxyproline and sulphated glycosaminoglycanes. These proven constituents of extracellular matrix were found to be released depending on the applied compressive strain.

Animals↗