Abstract:The γ-aminobutyric acid (GABA) shunt is a metabolic pathway that bypasses two successive steps of the tricarboxylic acid cycle (TCA cycle) in both prokaryotes and eukaryotes. In this pathway, the enzymes involved in GABA catabolism are GABA transaminase (GABASE), which converts GABA to succinic semialdhyde, and succinic-semialdehyde dehydrogenase (SSADH), which oxidizes succinic semialdhyde to succinate. In this report, we characterized gabT and gabD genes cloned from B.thuringiensis strain G03 isolated in China. Both gabT gene, 1440 bp encoding a protein with 52.6 kD, and gabD gene, 1449 bp encoding a protein with 52.2 kD were expressed in E.coli BL21(DE3) strain, and their products were purified by affinity chromatography. By enzyme assay,GabT protein showed the activity of GABASE, while GabD protein exhibited SSADH activity. The amino acid sequences of two proteins, GABASE and SSADH, showed significant identity with those in the B.cereus group, but their similarity score between G03 and B.subtilis 168 was lower, only 58% and 51%, respectively. In the present study, we provided evidence to identify the genes involved in GABA metabolism, and it will be helpful for further assessment the biological function(s) and regulation mechanism of GABA shunt in B.thuringiensis.

Abstract:To produce antibodies capable of neutralizing botulinum neurotoxin type B (BoNT/B), We cloned the carboxy-terminal end of Hc containing the major determinants responsible for specific toxin,induced and purifed. The heavy-chain and kappa light-chain variable region gene repertoire of immunoglobulin were amplified individually from the spleen cell mRNA by RT-PCR and joined as a single-chain Fv (scFv) DNA fragment. These fragment were cloned into the phagemid pCANTAB5E and the phage display library was constructed. Results showed that the high affinity scFv was obtained after 4 rounds of panning, with its DNA sequence conforming to that of mouse antibody.

Abstract:Two lipase active fractions Lip1 and Lip2 were purified from the cell extract of Rhizopus chinensis CCTCCM201021.Both gave a single band on SDS-PAGE after using ammonium sulfate precipitation、Phenyl-Sepharose FF、DEAE-Sepharose FF and Sephadex G100 gel filtration chromatographies. The molecular masses of two lipases were 59.2kD and 39.4kD respectively. Lip1 and Lip2 showed optimal pH at 8.0 and 8.5 and their optimal temperatures were 40℃ and 35℃ respectively. The substrate specificity of the two lipases was obviously different. Lip1 was more specific to long chain fatty acid of p-nitrophenyl esters while Lip2 had a preference for the hydrolysis of short chain fatty acid of p-nitrophenyl esters. Lip1 had 1,3-position specificity for triacylglycerols hydrolysis while Lip2 had nonspecific position. Both lipases were stimulated by Ca²⁺、Mg²⁺ while SDS had strong inhibition on their activities. Lip1 and Lip2 had good stability in cyclohexane、hexane、heptane and isooctane(30% V/V).

Abstract:14 strains were isolated from pits sludge by anaerobic cultivation, two Clostridium butyricum strains were identified by physiological and biochemical experiments and the sequence analysis of 16S rDNA. The physiological characteristics and security of Clostridium butyricum B1 were studied, in vitro research indicated it was tolerant against low pH, bile concentration and antibiotics and has antagonism effects against pathogens.

Abstract:A biotrickling filter which was packed with plastic striplike material, elasticity threadlike material and sponges was designed to remove odour from garbage compressing station.The air flow was increased to 8000 m³/h to obtain EBRTs of 2.5s. NH₃ elimination capacities were observed. The liquid medium recirculated resulted in an increase of the elimination capacity from η=75% to η= 90%. The biomass and microorganism activity on the filling materials were tested, the results shown that microorganism quantity and activity decreased in the order of plastic striplike material > elasticity threadlike material > sponges. Finally how to improve the treatment efficiency from the aspects of technics and types of film carriers were discussed.

Abstract:A bacterial strain that produces glutathione(GSH) was screened from vegetable garden soil based on a polymerase chain reaction (PCR) protocol which could rapidly and specifically identify the gshB gene coding for GSH synthetase. Phylogenetic analysis based on 16S rDNA revealed that this strain belongs to L. lactis ssp. cremoris. Scanning electron microscope analysis indiczated that its morphology characteristics were most consistent with Lactococcus lactis. So we suggested that the strain belonged to L. lactis ssp. cremoris and named L. lactis CCSYU10100. The intracellular level of GSH in stationary-phase cells of strain CCSYU10100 grown aerobically in chemically-defined medium was 2.39±0.21 nmol/mg protein. Other than GSH, cysteinylglycine was identified by HPLC. The partial gshB gene sequence and the deduced amino acid sequence of strain CCSYU10100 were highly similar with that of Pseudomonas among different microorganisms, which indicated that strain CCSYU10100 probably possesses independent gshB gene. Noticeably, the GC content of the partial gshB gene of strain CCSYU10100 was 61.55%, and the GC content of whole genome of L. lactis is 35%-40%, which seggusted that the gshB gene might not be an inherent part of strain CCSYU10100.Considering Pseudomonas fluorescens exists in the soil widely, it was speculated that the origin of gshB gene of strain CCSYU10100 was from Pseudomonas fluorescens by lateral gene transfer. The deduced amino acid sequence of strain CCSYU10100 contained two conserved domain, i.e., prokaryotic GSH synthetase, N-terminal domain (2^nd～58^th amino acid residues) (identities 44.5%) and rokaryotic GSH synthetase, ATP-binding domain(61^st～221^st amino acid residues) (identities 93.4%). To our knowledge, this is the first report showing gshB gene exists in Lactococcus lactis, even in Gram-positive bacteria.

Abstract:A biosurfactant-producing strain (S₆) was isolated from oil-containing wastewater in oxidation ditch and identified as Pseudomonas aeruginosa based on physiological and biochemical experiments and 16S rDNA sequence analysis. Infrared spectrum analysis revealed that S₆ produced glucolipid in the process of metabolism. It was observed that S₆ decreased the surface tension of water from 72 mN/m to 33.9 mN/m with the critical micelle concentration (CMC) of 50mg/L. The measurement of oil displacement and surface tension demonstrated that the fermented liquid had stable surface activity at varying range of salinity, pH, amount of dissolved oxygen. The optimal culture condition was obtained through orthogonal experiment: glucose 10g/L, urea 5g/L, KH₂PO₄ 1g/L, liquor of microelement 2mL, pH 8.0, water 1000mL; and the biosurfactant production under optimal culture condition was 0.173g/L.

Abstract:Optimum technique of isolation and purification of Coprinus comatus polysaccharides was investigated by orthogonal tests. DEAE-Sepharose F. F. and gel-filtration chromatography were used to separate and purify the Coprinus comatus polysaccharides, and get the homogeneous polysaccharide CC30w-1.The structure of CC30w-1 was studied with chemical and spectral methods. The results showed that the optimum conditions for the isolation was as follows： times of extraction 3, time 1.5h, temperature 95℃, material to water ratio 1∶12.The optimum deproteinization condition is that crude polysaccharide solute in the mixed solvent of chloroform and n-butanol (3∶1) whose volume 3 times that of the former, the reaction time and times of purification were 20 minutes and 7 times, respectively. CC30w-1 molecular weight was estimated to be 1.94×10⁴ by HPLC. HPAEC showed CC30w-1 was composed of fucose and galactose in molar ratios of 1∶4.02. Methylation analysis and NMR data showed that it possess a pentasaccharide repeating unit have a backbone of (1→6)-linked galactose residues, and is substituted at 2-position by fucose residue.

Abstract:The potential of a Sinorhizobium fredii strain producing a copolymer polyhydroxyalkanoate (PHA) from glucose and sodium decanoate substrates was studied in this paper. Using orthogonal design in a flask-shaker culture system, the culture medium, some culture conditions and vital regulation conditions for polymer synthesis were optimized. These optimized results were applied into further studies in two-stage fed-batch with a 10L fermentor. The whole culture process consisted of two stages, that is, the cell growth and the copolymer production. The first stage was for the cell growing to a desired biomass and the second was for the copolymer synthesis. For producing PHA polymers, the selected 8 mM sodium decanoate was added into the broth by adopting a two-step adding method for avoiding of foaming when the biomass had approached 28.5g/L dry cell. The maximum P (HB-HH) production could be 17.55g/L with a monomer ratio of 79.4% (W/W) 3-HB and 20.6% (W/W) 3-HH. The molecule constitute of the copolymer is poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) ［P (HB-HH)］ and its molecular weight (MW) is 1.4×10⁵D. The results demonstrated that the employed S.fredii strain could be a potential candidate for industrial production of the copolymer. The fermentation parameters acquired in the experimental system offered some valuable references for studying large-scale production of the copolymer.

Abstract:The producing condition and properties of chitinase secreted by Nomuraea rileyi strain CQ031021 were studied. The optimal conditions for the strain to produce chitinase are 6 days of 28℃ with the initial pH 6.0, and the liquid medium containing 2.0% glucose as its carbon source and 0.6% peptone plus 0.6% beef extract as nitrogen source after inoculating dosage 2mL suspension of conidia (1×10⁷/mL). The optimal temperature and pH for enzyme activity is 50℃ and 6.0, respectively, while the activity can be enhanced by Tween-80 and inhibited by SDS. The enzyme activity is stable under 40℃ and in pH range of 5.5～6.5.

Abstract:Four mating types of basidispores in 3 strains of Pleurotus ostreatus were determined using a nuclear migration test and an OWE-SOJ technique. The nuclear migration test showed that nuclear migration only occurred in A=B≠ mating interaction but did not in A≠B= interaction. In OWE-SOJ test, a fluffy colony without clamp connection formed in the A=B≠ mating interaction where nuclear migration occurs, while a barrage colony without clamp connection formed in A≠B= interaction where nuclear migration do not occur. The two colonies could be distinguished clearly each other. The result of the nuclear migration test is consistent with that of OWE-SOJ, which could be corroborated each other. Therefore it is feasible to apply the two to accurate determination of mating types in Pleurotus ostreatus.

Abstract:Streptomyces sp. X-435 isolated from a soil sample collected in the suburbs of Beijing was proved to be a produce Virginiamycin. To improve the productivity of the Virginiamycin of Streptomyces sp. X-435,the spores of strain X-435 were treated with UV. The three types of colony,strawhat, wrinkled, blad, were isolated on Gaose's medium plates after mutation. Among them, the colonies of strawhat type exhibited positive mutation and were picked up as objects of screening. After five generation of mutation, the mutant F5-25-u-28 was selected which potency of Virginiamycin was about 20times higher than that of the beginning strain by flask fermentation and was also genetic stable.

Abstract:The cell attachment protein gene of the S1 segment of reovirus BYD was cloned into prokaryotic expression vector, pET-28a(+). The resulting pET-28a(+)-S1 was transformed into E.coli BL21(DE3) for analyzing whether the constructed vector could express inferred recombinant σ1 and characterizing the antigenicity of the recombinant σ1.SDS-PAGE showed the constructed vector expressed a protein around 53 kilodalton, which was consistent with the deduced molecular weight of the recombinant σ1.And immunoblot assays demonstrated that the recombinant σ1 had good antigenicity and specificity, and therefore it should have great potential in developing diagnosis reagent.

Abstract:A heavy metals hyperresistant strain Bacillus cereus, HQ-1 with high resistance to Pb、Zn、Co、Cu、Cd and Ag was isolated from a lead-zinc mine. Minimal inhibitory concentration of Cd²⁺ for the bacterium was 1200 mg/L higher than others. The biosorption isotherms of cadmium on cells of Bacillus cereus strain HQ-1was investigated and compared with silver. It has a strong adsorptive capacity to the two metal ions．Its adsorption behavior could be described by either the Langmuir adsorption equation or the Freundlich adsorption equation. The adsorption mechanism of Cd²⁺ and Ag⁺ were studied by FTIR spectroscopic analysis and energy dispersive X-ray spectroscopy. We also simply located the resistant gene on the plasmid of the strain. The results of this study indicate that this Bacillus cereus strain has an excellent potential for biosorption and bioremediation of heavy metals pollution.

Abstract:The isolated 24 strains-producing hydantoinase & carbamoylase were first identified by Biolog microbial identification system and 16S rDNA sequence analysis. The results suggested that the hydantoinase & carbamoyalse-producing bacteria belonged to Bacillus, Geobacillus, Brevibacillus, Aneurinibacillus, Microbacterium, Pseudomonas, Kurthia and Empedobacter, and so on. Especially, Kurthia and Empedobacter were new hydantoinase & carbamoylase-producing genera. Furthuremore, it was found that D-hydatoinase & carbamoyalse-producing bacteria belonged to Pseudomonas and Agrobacterium, while most of L-hydantoinase & carbamoyalse-producing bacterial belonged to Bacillus，Geobacillus and Microbacterium. The distribution feature of D-hydantoinase & carbamoyalse-producing bacteria and L-hydantoinase & carbamoyalse-producing bacteria showed some genera tendency. This research work will provide the biomaterial of different hydantoinase and carbamoylase and contribute to study the structure and function, molecular evolution of the two enzymes.

Abstract:Raman tweezers, a novel technique that combines Raman spectroscopy with optical tweezers, has the potential to become an effective tool for analysis single cell suspended in solution. To real-time investigate the biochemical proceedings in single cell, a Raman tweezers setup was used to trap a single instant active dry yeast cell and to acquire the real-time Raman spectrum of the trapping cell from dormancy to activation cultured in 2.0% (W/V) glucose. During the activating process, the intensity of bands derived from protein or lipids, such as 1000 cm^-1，1445 cm^-1，1655 cm^-1, were not changed evidently. The intensity of the 1603 cm^-1 band, which was considered as the signature of metabolic activity, was not changed evidently too. But the bands at 531cm^-1，652cm^-1，1053cm^-1，1364 cm^-1, which were assigned to trehalose, or glucose-base carbohydrates, were increased as the cell grew. This phenomenon, occurred in seven individual experiments of ten repeats using a batch of instant active yeast cells as tested cells, but did not occur when other batches of instant active yeast cells were tested. This peculiar process of adaptation was recorded by real-time Raman spectrum and the result indicates Raman tweezers is an effective tool for single-cell analysis.

Abstract:The purpose of this paper is to examine the pH adaptability range of two yeasts from our laboratory, and applied turbidimetry and Bradford methods to examine growth of Trichosporon asahii XJU-1 and Rhodotorula mucilaginosa XJU-1.It is shown that Trichosporon asahii XJU-1 grown between pH2.0 and pH13.0 and optimum pH is 8.0, whereas Rhodotorula mucilaginosa XJU-1 grown between pH3.0 and 12.0，optimum pH is 8.5. When turbidimetry was applied, it produced consensus results between pH4.0 and 10.0 with Bradford method. At the same time, produced senior distorted at pH<4.0 and pH≥10.0.Bradford method indirect determined total soluble protein of Trichosporon asahii XJU-1 is 290μg/mL at optimum pH8.0, Rhodotorula mucilaginosa XJU-1 is 164μg/mL at optimum pH8.5, respectively. Compared with turbidimetry, Bradford method embodied its accuracy in whole pH ranges. It is concluded that both two tested yeasts have wider pH tolerant ranges and can be two new members of alkali-tolerant yeast family.

Abstract:A marine bacterium MWYL1, originally isolated from the roots of Spartina anglica growing in a salt marsh near seaside, was identified as a member of the genus of Marinomonas via morphology characterization、physiological test and 16S rDNA sequencing and Blast analysis. The strain was short, rod, gram-negative, grew aerobically and optimally at 28℃. The analysis of 16S rDNA sequence suggests that the sequence similarity values are 97% and 95% with Marinomonas pontica and Marinomonas dokdonensis, respectively. One fosmid clone producing melanin was directly isolated by plating from the genomic library of Marinomonas MWYL1.The novel functional gene cluster involved in melanin biosynthesis was screened after subcloning and sequencing of the 14kb insert in pUC18, further more, the putative functional genes was preminary analyzed using bioinformatics.

Abstract:Effect of p-nitrophenol shock on microbial populations and sludge activity in UASB reactor was studied by DGGE-PCR of 16S rDNA fragments and detection of COD removing and biogas yield. The results showed that p-nitrophenol seriously inhibited the sludge activity, resulting in the drop of biogas and COD removing rate. The 40mg/L p-nitrophenol had more inhibition than 20mg/L p-nitrophenol. It would take 27 and 16 days respectively for reactor to recover after 40mg/L and 20mg/L p-nitrophenol shock. The diversity of eubacteria and methanogens were also effected by the p-nitrophenol shock. The variation of eubacteria was more than that of methanogens after p-nitrophenol shock. The drop of biogas was mainly related to the variation of Methanosaeta sp. and Methanomicrobia sp. after p-nitrophenol shock. Among the eubacteria the population of Chloroflexi sp.、Bacteroide sp. and Anaerovibrio sp. decreased greatly after p-nitrophenol shock. And more, the Rheinheimera sp disappeared after 40mg/L p-NP treatment. But the Flavobacteria sp. appeared after p-nitrophenol shock, which was probably related to the degradation of p-NP.

Abstract:Using gene recombinant techniques, two chimeric operons containing rhaSR promoter, RhaR gene, and reporter gene gst (glutathione S-transferase )were constructed, and each was inserted into the E.coli expression vector pALEX to form pALEX-PR1 and pALEX-PR2.The pALEX-PR2 contained a native SD sequence in the upstream region of rhaR, while the pALEX-PR1 contained an enhanced SD sequence. Two plasmids were then transformed into E.coli BL21 (DE3). The reporter gene gst within both chimeric operons expressed 4 to 5 folds higher with L-rhamnose induction than without the induction. Under the induction of L-rhamnose, the GST expression of the pALEX-PR1 was 3.14 folds than the pALEX-PR2.Our results suggested that the expression of gst was positively regulated by the induction of L-rhamnose and the RhaR expressed from the same chimeric operon. Furthermore, SDS-PAGE results showed that GST accounted for about 5.41%(W/W) of the total soluble proteins of the E. coli culture. An average of 3.0 mg purified GST was obtained from 1 L culture. The results of enzyme activity analysis showed that the GST expressed by reporter gene gst of our chimeric operons kept the correct configuration and high activity.

Abstract:Microbial hydroxylation of androst-4-ene-3,17-dione(4AD) was investigated in this study.The hydroxylated product of 4AD,11α-hydroxyandrost-4-ene-3,17-dione (11α-OH-4AD) is valuable intermediate in the synthesis of pharmaceuticals. 30 different species and strains of fungi were screened for their biotransformation ability. It was found that Beauveria bassiana QY_{2A strain significantly showed the high 11α-hydroxyalate efficiency and transformate 4AD to the target product 11α-OH-4AD which had been purified and identified.The hydroxylation condition of 4AD by B. bassiana QY2A was studied.The optimum transformation conditions were obtained: the initial pH value of 6.0, temperature 28℃, 180r/min, the transformation time 60h, the concentration of solution and substrate were 2.5% and 2.5g/L respectively. Then the conversion yield of 11α-OH-4AD was 65%, increased by 51.2%.}

Abstract:To study the incidence rate of Salmonella, seven strains bacteria-specific phages as a group were used for identification of Salmonella species. All of the 60 bacteria (for test) were inoculated in Luria Bertani (LB) broth, cultivated in 37℃ for 2 hours, 6 hours, 10 hours and≥18 hours respectively, then tested on agar plate and in LB broth, and made micro-biochemical identification through automatic biochemistry analyzer, to confer the most suitable inoculated hours for test bacteria. The result showed that, tested on agar plate, the positive rates of Salmonella cultivated for 2h, 6h, 10h and ≥18h were 91.1%, 93.3%, 97.8% and 88.9% respectively; while tested in LB broth, a reasonable result was showed in the 10h-inoculated tested bacteria. It was resemble to test with food samples bought from some markets in Nanjing. It provided theoretical evidence for performing detection Salmonella species in food with real time, speed, selective and specific.

Abstract:The phytase was extracted from solid state leavening of Trichoderma Viride LH374. The crude product was purified by (NH₄)₂SO₄ precipitation, gel filtration and ion exchange chromatography. The purified phyatse was 13.3 times of the raw products, and the extraction ration was 27.1%. The study on the enzymology of phyatse showed that the optimal temperature and pH were 55℃ and 6.0, respectively. The Km value of the phytase was 0.15mmol/L.

Abstract:The acid-and bile-tolerant, the transit tolerance, the ability to adhere to human intestinal cells and the ability to assimilate cholesterol of seven lactic acid bacteria and Bifidobacteria strains were studied by an in vitro method. The abilities to adhere to CCL-187 cells were different between all strains tested(P<0.05) and high attachment was observed with Enterococcus faecium M1.All strains tested were considered intrinsically resistant because the viability of them can remain >95% when incubated in simulated gastric and small intestinal juice for 3h and 4h respectively. All strains have the ability to grow in the presence of bile, and the ability varied among the strains. The cholesterol assimilated ranged from 25% to 54%. Enterococcus faecium A30, A31 and Lactobacillus acidophilus A878, PB1, which assimilated more than 40% cholesterol from circumstances, can be good hypocholesterolemic candidate strains for functional food.

Abstract:Based on a β-tubulin gene EST sequence from Trichoderma harzianum mycelium cDNA library, a 1.74kb β-tubulin gene coding region, together with a 1.5kb 5′-flanking region and a 1.0kb 3′-flanking region was cloned from T. harzianum by IPCR methods. The gene encodes a 446 amino acid polypeptide, which shows a high degree of homology with other fungal β-tubulins. A three-dimensional model of the T. harzianum β-tubulin was built based on the crystal structure of bovine tubulin using homology modeling method. Tertiary protein structure of the β-tubulin should provide a basis for understanding a significant body of research on tubulin′s properties and interactions with antimicrotubule agents.

Abstract:1.6 kb Δ⁴-desaturase gene(FAD4)was amplified by PCR using plasmid pGEM-TFAD4 as template. The fragment was subcloned into the HindⅢ/XbaⅠrestriction site of pYES2.0 vector. Recombinant plasmid pYFAD4 was transformed into Saccharomyces cerevisiae strain INVScl for expression.It was found to exhibit Δ⁴-fatty acid desaturase activity in the recombinant S.cerevisiae YFAD4 in the presence of exogenous fatty acid substrate docosapentaenoic acid(100μmol/L)under introduction of GAL1.Expression of the FAD4 under appropriate media and temperature conditions led to the production of DHA and it reached 41.13％ of the total yeast fatty acid by GC detection.It was suggested that the protein encoded by FAD4 could specifically catalyze DPA into DHA.

Abstract:An oligonucleotide primer pair was designed and synthesized after comparison and homological analysis of rDNA ITS sequences among Phytophthora sojae, its related Phytophthora species，and allied fungal and bacterial species from GenBank. PCR amplifications were carried out for 140 isolates including Phytophthora sojae.It showed that only isolates of Phytophthora sojae can be amplified and a special fragment of 288bp were produced by the primers. These primers were used to detect Phytophthora sojae in pure culture，inoculated diseased soybean plants， and inoculated soil samples. The detection protocol has good sensitivity to diseased tissues.

Abstract:Urate oxidase belonging to the purine degradation pathway, catalyzes the oxidation of uric acid to allantoin. It′s useful for enzymatic determination of urate in clinical analysis. It can also be used as protein drug for treatment of gout, hyperuricemia and tumor lysis syndrome (TLS). The source, enzyme property, genetic engineering and application of uricase were reviewed in this paper.

Abstract:As a result of various anthropogenic activities, natural saline environment polluted by contaminants and the environment polluted by both contaminants and salts frequently occur. In these saline environments, non-halophilic microorganisms have ineffective bioremediation, even lose the function of bioremediation. Halophilic microorganisms are able to thrive in high salt conditions. It is obvious that the halophilic degraders are more useful for the bioremediation of contaminated saline environments and may be potentially used in a wide of application. The objective of this review is to summarize the research progresses of halophiles in the biodegradation of petroleum hydrocarbons, aromatic hydrocarbon ramifications, and organophosphorous.

Abstract:The fungal genetic transformation is an essential method for the research of functional genes. The latest advance in fungal genetic transformation system was reviewed in this paper, mainly including transformation methods and their advantages and disadvantages, types of selective markers, separation methods of the tagged gene and the application of the transformation system.

Abstract:Brucella organisms are facultative intracellular bacteria capable of surviving inside professional and non-professional phagocytes. Upon cell contact the bacteria is internalized via receptor molecules. Once inside cells, Brucella localizes in early phagosomes, where it avoids fusion with late endosomes and lysosomes. Then, the bacterium redirects its trafficking to autophagosomes and finally reaches the endoplasmic reticulum, the replicating niche. Once inside the endoplasmic reticulum, Brucella extensively replicates without restricting basic cellular functions or inducing damage to cells. Invasion, intracellular trafficking and replication of Brucella organisms in professional and non-professional phagocytes and the molecular determinants involving Brucella intracellular life are reviewed in this article.

Abstract:Microbiology is an important, fundamental and obligatory course in contemporary life science. This article introduces that teaching group of microbiology in Nankai University realizes transformation of teaching center, fully embodies the modernization of teaching notion and gives full play to students′ main effect practically by adhering to teaching reform as center, optimizing teaching method as measure, communicating in and after class and using multi-media and teaching web. Therefore, teaching system is established to adapt to modern teaching notion and eventually microbiology course becomes a cultivation platform to foster elites with both solid fundamental theory and innovating mind.

Abstract:As a major ingredient of high medical college education, experiment practice plays an important role for medical students in integrating the medical theory with practice, training their capability of analyzing and thinking to definitively solute the problems in their study. Microbiology experiment is characterized with strong comprehensiveness and manipulation, which requests the students both aseptic operation and consciousness of bio-safety. To promote the student′s comprehensive diathesis and to improve the experiment teaching quality, several common problems of students in medical microbiology experiment practice were analyzed and discussed in this article.

Abstract:To establish a simple，sensitive，accurate and rapid detection method for bifidobacteria. Molecular beacon probe and primers were designed and sythesized according to the conserved gene of Bifidobacterium in GenBank，and then reaction parameters of real-time PCR were optimized. For Real-Time PCR and molecular beacon detection for bifidobacteria, the intra-assay and inter-assay coefficient of variation were both less than 5%, indicating good reproducibility of this method, and no non-specific amplification was observed either. Compared with conventional PCR, this method is more sensitive and faster, and the detection limit was 5.7fg/PCR for bifidobacteria DNA. A linear standard curve was obtained between 2×10⁸CFU/mL and 2×10⁴CFU/mL (R>0.97). The method of Real-time PCR and molecular beacon detection for bifodobacteria has many advantages, such as being sensitive, specific, simple and fast, and this method can be used in situ detection of bifidobacteria quantitatively.

Abstract:Based on Staphylococcus aureus 16S rRNA gene sequences, sequence comparisons have been applied to design a kind of stem-loop structured oligonucleotide probes whose loop sequence mismatched other bacterial strains 16S rRNA gene sequences in more than two positions with high specificity and sensitivity. According to the principle of molecular beacon technology and enzyme linked immunosorbent assay, a method has been evaluated using immobilized stem-loop structured oligonucleotides probe as the conformation switch which is applied to enzymatic detection of nucleic acid targets. As its specificity has been strengthened, the system is able to successfully eliminate false-positive result that is mismatch in an oligonucleotide. Employing a microtiter assay format, 4ng of S. aureus 16S rRNA could be detected at least. This approach is more sensitive than other conventional method at least one order of magnitude.

Abstract:The plasmid pTE-okm, which was obtained by inserting the fluorescence gene eyfp into the plasposon pTnMod-okm, transferred from E.coli S17-1 into Shewanella decolorationis S12 by conjugation. pTE-okm could not replicate in Shewanella decolorationis S12 because of its narrow-host-range replication origin, but the minitransposon within pTE-okm could transpose to the chromosome of Shewanella decolorationis S12 and made it survive the LB medium with kanamycin and rifampicin. The clones which expressed fluorescence gene eyfp were screened from all the clones on the selected plates under fluorescent microscope and it was make sure that pTE-okm didn't exist in these clones. S12-40, which had the the same growth rate and decoloration ability with the original strain S12-1, was picked out within these clones. S12-40 expressed EYFP stably even after successive re-culturing for 20 times (8h/time). The construction of S12-40 laid a foundation for the study on the ecological performance of Shewanella decolorationis S12.

Abstract:Sands of different depth were sampled from Constructed Rapid Infiltrition system(CRI) in Shenzhen, and they were analyzed by Denaturing Gradient Gel Electrophoresis(DGGE). The profile of DGGE showed that, the microbial community decreased from top to bottom and in the upper 30cm part, CRI was rich in microbial community about 18 species which played some important roles in the biodegradation of COD, such as Firmicutes、alpha proteobacterium and so on; but less than 12 species existed below 40cm, such as Acidovorax sp.、Nitrospira sp.、Clostridium sp., this indicated that there were some nitrification phenomenon and anaerobic microenvironment in the lower part of CRI. The microbial diversity was prominent different spatially. The results showed the microbial community distribution and were certain meaningful to the stabilization and improvement of CRI.

Abstract:To screen the identifier of B.thuringiensis, genomic DNA of B.thuringiensis, B.cereus and control strains were amplificated by ERIC-PCR. Fragments amplified from B.thuringiensis were retrieved, cloned，marked by α-³²P, dot blot and southern hybridized with genomic DNA of these strains. It shows that every strains of B. thuringiensis can be amplified to produce a 250bp fragment, and both B.thuringiensis and B.cereus can be amplified to produce a 600bp fragment. Hybridization result between 569bp probe and genomic DNA of B.thuringiensis shows a high specificity. Discrimination and genetic relationship between B.thuringiensis and B.cereus can be revealed by ERIC fingerprint. This research indicates that ERIC-PCR technique is a practical method in the detection and characterization of B.thuringiensis and B.cereus.

Abstract:It is important for identifying, tracking and preventing pathogenic microorganism's outbreak in the epidemiology of infectious diseases. Establish a useful simple method for typing and identifying pathogenic microorganisms is necessary. In 1998, Multilocus sequence typing was proposed which is a nucleotide sequence based approach.It combined developments in high-throughput sequencing techniques with established population genetics.This typing method is a portable and reproducible typing system which can track and investigate pathogenic microbes distributed condition over the global by the internet.Now, MLST schemes have been developed for a variety of prokaryotic and some eukaryotic pathogens (like some fungus).In this paper, the theory of MLST and its application on some pathogenic microbes typing and identifying were introduced.

Abstract:Enterobacter sakazakii was an emerging food-borne pathogen associated with meningitis, sepsis and necrotizing enterocolitis, especially in neonates with high potential danger. The conventional detection methods were time-consuming and operated arduously, sometimes gave ambiguous signals. Increasing of reports showed the infant formula was the main infection vehicle. Consequently, it was important that improvement of methods for earlier detection and confirmation of presumptive E.sakazakii in infant formula to control and guard against the epidemic diseases due to E.sakazakii. In this study, PCR assay was developed based on ompA and α-1, 4-glusidase genes. Expected fragments(469 bp and 673 bp)were produced from 8 strains of E. sakazakii including E.sakazakii ATCC51329 after PCR amplification, but not from 70 strains of other bacteria. The sensitivity is 10¹ cfu/mL and 10² cfu/mL by signal-PCR using ESSF-ESSR and ESF-ESR as primers respectively in pure culture, sensitivity of dual-PCR is 10³ cfu/mL. Detection limit in artificially contaminated infant formula is 10² cfu/mL and 10³ cfu/mL by single-PCR with ESF-ESR and ESSF-ESSR respectively. To investigate whether the presence of other bacteria in infant formula have any effect on the sensitivity of PCR, two sets of experiment were designed. Different levels of other bacterial do not affect the PCR detection limit, which indicates the primers are species-specific for E.sakazakii. The investigation of actual samples shows that PCR assay is consistent with FDA standard method. The new method developed in the study can be used widely to detect the presence of E.sakazakii in infant formula with higher sensitivity and specificity than conventional methods.

Abstract:Proteolytic degradation has been a severe problem when Pichia pastoris is employed to express recombinant proteins.One alternative method to circumvent this problem is to construct protease gene disruptant. However, the main study of gene disruption is focused on nonrecombinant Pichia pastoris rather than recombinant strain. In our study, we established two different methods to directly disrupt PRC1 and KEX1 gene in recombinant Pichia pastoris. On the basis of this, we further discussed and compared the application and advantages of both methods.

Abstract:A new procedure for virus concentration from water samples has been developed. This method called calcium flocculation-citrate buffer method involves virus flocculation formed by 1 mol/L Ca²⁺ solution and its flocculating agent, virus release with sodium citrate dissolution (0.3 mol/L, pH5.0), and virus reconcentration by ultrafiltration. It can concentrate the viruses 10000 times easily. Seeding experiments showed that it could recover f₂ phage in an average rate of 96%, and 100% for the vaccine poliovirus type 1 (PV₁), which was significant higher than the current positive charged filter method (P<0.05). The established approach is rapid, simple and effective.