To optimize the electroporation system in Dunaliella salina (D. salina), we studied the effects of growth phase of cells, electric parameters, electroporation buffer and concentration of plasmid on transformation efficiency. The results showed that a transformation efficiency of 1.81 per thousand was achieved in D. salina cells at mid-log growth phase electroporated with plasmid (DCA-bar) 10 microg/mL, voltage 0.8 kV and capacitance 25 microF. However, when glycerol was added to electroporation buffer at a final concentration of 0.4 mol/L, the transformation efficiency was increased up to 2.03 per thousand. Additionally, transformation was done with plasmids DCA-bar, NR-bar, pUomega-bar respectively, under above optimum conditions, and similar transformation efficiencies were obtained. The findings indicate that an efficient and stable system of electroporation in D. salina has been developed, providing a powerful tool for the transgenic research of D. salina.
Chlorophyta ; cytology ; genetics ; Culture Media ; Electroporation ; Organisms, Genetically Modified ; genetics ; Transformation, Genetic ; genetics

Trehalose, a compatible solute, is widely used in food, cosmetics, pharmaceutical products and organ transplantation. Nowadays, trehalose is mostly produced by enzymatic synthesis with many secondary products and lowpurity. In this study, high amount of trehalose was produced by recombinant E. ccli fermentation. First, a bifunctional trehalose gene TPSP was amplified from genome of C. hutchinscoii. Second, an expression vector pTac-HisA containing TPSP was constructed and transformed into the host E. coli. Expression of this bifunctional enzyme-TPSP converted glucose to trehalose. The result suggested that TPSP from C. hutchinsonji has been successfully expressed in E. ccoi. High amount of extracellular trehalose generated from glucose by whole-cell catalysis and After optimization, the production of trehalose in shake flasks was improved to 1.2 g/L and the relative conversion rate reached 21%. The production in bioreactor reached 13.3 g/L and the relative conversion rate reached 48.6%. It is the first time to realize the functional expression of the bifunctional enzyme-TPSP of C. hutchinsonii in E. coli and achieved the conversion form glucose to trehalose. This study laid a foundation for industrial large-scale production of trehalose.
Bioreactors ; Catalysis ; Escherichia coli ; genetics ; Glucose ; Glucosyltransferases ; Industrial Microbiology ; Organisms, Genetically Modified ; Trehalose ; biosynthesis

Pyrroloquinoline quinone (PQQ), an important redox enzyme cofactor, has many physiological and biochemical functions, and is widely used in food, medicine, health and agriculture industry. In this study, PQQ production by recombinant Gluconobacter oxydans was investigated. First, to reduce the by-product of acetic acid, the recombinant strain G. oxydans T1 was constructed, in which the pyruvate decarboxylase (GOX1081) was knocked out. Then the pqqABCDE gene cluster and tldD gene were fused under the control of endogenous constitutive promoter P0169, to generate the recombinant strain G. oxydans T2. Finally, the medium composition and fermentation conditions were optimized. The biomass of G. oxydans T1 and G. oxydans T2 were increased by 43.02% and 38.76% respectively, and the PQQ production was 4.82 and 20.5 times higher than that of the wild strain, respectively. Furthermore, the carbon sources and culture conditions of G. oxydans T2 were optimized, resulting in a final PQQ yield of (51.32±0.899 7 mg/L), 345.6 times higher than that of the wild strain. In all, the biomass of G. oxydans and the yield of PQQ can be effectively increased by genetic engineering.
Fermentation ; Gene Knockout Techniques ; Gluconobacter oxydans ; genetics ; metabolism ; Industrial Microbiology ; methods ; Multigene Family ; genetics ; Organisms, Genetically Modified ; PQQ Cofactor ; biosynthesis ; genetics ; Promoter Regions, Genetic ; genetics

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Proliferation ; Interleukin-10 ; genetics ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Organisms, Genetically Modified ; Rats ; Rats, Sprague-Dawley ; Transfection

OBJECTIVETo study the expression of brain-derived neurotrophic factor (BDNF) gene modified bone marrow mesenchymal stem cells (MSC) in the cochlea of drug-deafened guinea pigs and its protection to spiral ganglion cells (SGC).

METHODSGuinea pigs deafened by subcutaneous injection of amikacin were randomly divided into two groups, BDNF gene modified bone marrow MSC were injected into the cochlea through fenestration of scala tympani in the experimental group, while artificial perilymphatic fluid were injected in the control group. Experimental animals were executed at 7 and 28 days post-operation. Expression of BDNF mRNA was examined by quantitate real time RT-PCR, histological images of cochlear sections were analyzed to calculate the cellular density of the SGC, and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) was used to identify the apoptotic neurons.

RESULTSThe BDNF expressive level in experimental group was higher than in the control group at 7 d and 28 d post-operation, whose differences were both statistically significant (P < 0.01). And, It showed a higher abundance of ganglion cell numbers, as well as a decreased apoptotic index in experimental group compared with the control group at 7 d and 28 d post-operation, whose differences were all statistically significant (P < 0.01).

CONCLUSIONBDNF gene modified MSC could maintain expression for at least 28 days after transplantation into cochlea of drug deafened guinea pigs, and protect SGC.

Animals ; Bone Marrow Cells ; metabolism ; Brain-Derived Neurotrophic Factor ; genetics ; pharmacology ; Deafness ; chemically induced ; therapy ; Guinea Pigs ; Mesenchymal Stromal Cells ; metabolism ; Organisms, Genetically Modified ; Spiral Ganglion ; drug effects

Recombinant plasmid pICG was constructed by replacing the internal fragment of a-acetohydroxyacid synthase (AHAS) gene (ILV2) with a copy of gamma-glutamylcysteine synthetase gene (GSH1) and copper chelatin gene (CUP1) from the industrial brewing yeast strain YSF31. YSF31 was transformed with plasmid pICG linearized by Kpn I and Pst I. A recombinant strain with high-glutathione and low-diacetyl production was selected. The results of fermentation in 100-L bioreactor showed that the lagering time of beer produced for recombinant strain T2 was shortened by 3 days and the shelf life of the beer was prolonged about 50%. It may be more acceptable for the commercial application, as it does not contain foreign DNA.
Acetolactate Synthase ; genetics ; metabolism ; Beer ; microbiology ; Cloning, Molecular ; Diacetyl ; metabolism ; Fermentation ; Gene Expression Regulation, Fungal ; Glutamate-Cysteine Ligase ; genetics ; metabolism ; Glutathione ; biosynthesis ; Metallothionein ; genetics ; metabolism ; Organisms, Genetically Modified ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism

In order to effectively cure hepatitis B virus (HBV), we studied on fusion protein HBscFv-IFNgamma, which was connected with single-chain Fv against HBV surface antigen(HBscFv) and gamma-interferon(IFNgamma) of being used in clinic against HBV. Adopting overlap PCR, the hbscfv and the ifngamma were connected into hbscfv-ifngamma. Then the pPICZalphaA/(hbscfv-ifngamma)(1,2,4) of multi-copy recombinant plasmid were constructed and transformed into Pichia pastoris x33. The engineering strain x4 was screened from transformed x33 and could secretively express HBscFv-IFNgamma. The preliminary verification indicates that HBscFv-IFNgamma has the bioactivity of HBscFv and IFNgamma by SDS-PAGE, Western blotting and ELISA. The supernatant of culturing X4 was purified by 14F7 affinity chromatography to HBscFv-IFNgamma with purity of 95%-98%. The HBscFv-IFNgamma is able to bind 27.9% HBV surface antigen (HBsAg) in the serum of HBV transgenic mice, which shows the antibody of HBscFv-IFNgamma has bioactivity in vivo. Therefore HBscFv-IFNgamma can shed light on the development of a new promising HBV-targeted drug.
Chromatography, Affinity ; Genetic Engineering ; Genetic Vectors ; Hepatitis B Antibodies ; biosynthesis ; genetics ; Hepatitis B Surface Antigens ; immunology ; Immunoglobulin Fragments ; genetics ; Immunoglobulin Variable Region ; genetics ; Interferon-gamma ; biosynthesis ; genetics ; Organisms, Genetically Modified ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

To construct the recombinant vector Pact-EGFP, the Act-1 core promoter region was amplified from the pUCm-T/Act-1 and subcloned into pEGFP-4.1 vector (derived from pEGFP-N1 with the removal of human cytomegalovirus immediate early promoter), by restriction enzymes Bgl II and Hind III. Transfection of Pact-EGFP vector into Vero cell by liposome indicated that Act-1 core promoter regulated the expression of EGFP gene in lower level in Vero cells. After Pact-EGFP microinjection into the gonad of Caenorhabditis elegans with pRF4 as a gene marker, green fluorescence was detected in the cortex, vice cortex and the pharyngeal of C. elegans. According to the locations, two different transgene lines were separated. The expression level of EGFP expressed in C. elegans was more than that in Vero cell. Some unique motifs might exist in Act-1 core promoter region of C. elegans, which was closely related to the expression level of EGFP. These results lay the foundation for the further research on gene function of parasitic nematodes using C. elegans.
Animals ; Caenorhabditis elegans ; genetics ; metabolism ; Cercopithecus aethiops ; Connexin 43 ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Organisms, Genetically Modified ; Peptide Fragments ; genetics ; Promoter Regions, Genetic ; genetics ; Transcription, Genetic ; Vero Cells

Construction of multifunctional pesticides-degrading genetically engineered microorganisms (GEMs) is increasing important in the bioremediation of various pesticides contaminants in environment. However, construction of genetically stable GEMs without any exogenous antibiotic resistance is thought to be one of the bottlenecks in GEMs construction. In this article, homologous recombination vectors with the recipient's 16S rDNA as homologous recombination directing sequence (HRDS) and sacB gene as double crossover recombinants positive selective marker were firstly constructed. The methyl parathion hydroalse gene (mpd) was inserted into the 16S rDNA site of the carbofuran degrading strain Sphingomonas sp. CDS-1 by homologous recombination single crossover in the level of about 3.7 x 10-(7) - 6.8 x 10(-7). Multifunctional pesticides-degrading GEMs with one or two mpd genes inserted into the chromosome without any antibiotic marker were successfully constructed. The homologous recombination events were confirmed by PCR and southern blot methods. The obtained GEMs were genetically stable and could degrade methyl parathion and carbofuran simultaneously. The insertion of mpd gene into rrn site did not have any significant effect on recipient' s physiological and original degrading characteristics. The methyl parathion hydrolase (MPH) was expressed at a relatively high level in the recombinants and the recombinant MPH specific activity in cell lysate was higher than that of original bacterium (DLL-1) in every growth phase tested. The highest recombinant MPH specific activity was 6.22 mu/tg. In this article, we describe a first attempt to use rRNA-encoding regions of Sphingomonas strains as target site for expression of exogenous MPH, and constructed multifunctional pesticides degrading GEMs, which are genetically stable and promising for developing bioremediation strategies for the decontamination of pesticides polluted soils.
Biodegradation, Environmental ; Carbofuran ; metabolism ; Environmental Pollutants ; metabolism ; Genetic Markers ; Insecticides ; metabolism ; Organisms, Genetically Modified ; genetics ; metabolism ; Phosphoric Monoester Hydrolases ; genetics ; metabolism ; RNA, Ribosomal, 16S ; genetics ; Recombination, Genetic ; Sphingomonas ; genetics ; metabolism

Iro system and temperature-sensitive hemagglutinin (Tsh) genes were identified by suppression subtractive hybridization (SSH) and selective capture of transcribed sequences (SCOTS). To get more insights in the distribution and the occurrence of the iroC and tsh genes, we examined 243 avian E. coli strains for the presences of the these genes. Among 243 avian E. coli isolates, iroC gene was present in 84.4% strains (205/243). Of the 205 iroC-positive isolates, iroC gene was found in 184 (89.8%), 18(8.8%) and 3 (1.5%) isolates with high, intermediate and low pathogenicity, respectively. Of the 167 tsh-positive isolates, tsh gene was detected in 146 (87.4%), 21 (12.6%) and 0 (0%) isolates with high, intermediate and low pathogenicity, respectively. Among tsh-positive isolates, 89.5 to 100% of the highly pathogenic isolates of O1, O2 or O78 serogroups had the tsh gene, while 53.3% of the highly pathogenic isolates of non-O1, O2 and O78 serogroups had the tsh gene (P<0.01). Suicide vectors for deletion of the iroBCDEN or tsh genes were constructed as follows. The 715-bp fragments of iroB and 603-bp fragment of the iroN were generated by PCR respectively. Both of these two fragments together with EGFP gene were cloned into pUC18, termed pUC18-iroBNEGFP. A resultant suicide vector containing the iroB-EGFP-iroN fragment was obtained and named pMEG375-iroBNEGFP. Similarly, both of the 685-bp fragment of tshF and the 692-bp fragment of the tshR together with gentamycin gene were cloned into pUC18, resulting in pUC18-tshFRGm. A resultant suicide vector containing the tshF-Gm-tshR fragment was named pMEG375-tshFRGm. Mutant derivatives of strain E037 were generated by allelic replacements and were named E037(Deltairo), E037(Deltatsh) and E037(DeltairoDeltatsh). The 50% lethal dose (LD50) of E037, E037(Deltairo), E037(Deltatsh) and E037(DeltairoDeltatsh) in commercial day-old chickens experimentally inoculated via intratrachea were determined to be 10(5.6), 10(8.4), 10(9.0) and 10(9.5)CFU, respectively. In the chicken challenging model, the mutants were tested to determine the individual role of this system for virulence and persistence in chickens. The result suggested that Iro system and Tsh were important in the pathogenicity of APEC.
Adhesins, Escherichia coli ; genetics ; Animals ; Chickens ; Escherichia coli ; genetics ; pathogenicity ; Escherichia coli Infections ; microbiology ; veterinary ; Genes, Bacterial ; genetics ; Mutation ; Nucleic Acid Hybridization ; methods ; Organisms, Genetically Modified ; Poultry Diseases ; microbiology ; Transformation, Genetic ; Virulence Factors ; genetics