A MEMS based amperometric detector for E. coli bacteria using self-assembled monolayers.

We developed a system for amperometric detection of Escherichia coli (E. coli) based on the integration of microelectromechanical systems (MEMS), self-assembled monolayers (SAMS), DNA hybridization, and enzyme amplification. Using MEMS technology, a detector array was fabricated which has multiple electrodes deposited on a Si wafer and was fully reusable. Using SAMs, a monolayer of the protein streptavidin was immobilized on the working electrode (Au) surface to capture rRNA from E. coli. Three different approaches can be used to immobilize streptavidin onto Au, direct adsorption of the protein on bare Au, binding the protein to a biotinylated thiol SAM on Au, and binding the protein to a biotinylated disulfide monolayer on Au. The biotinylated thiol approach yielded the best results. High specificity for E. coli was achieved using ssDNA–rRNA hybridization and high sensitivity was achieved using enzymatic amplification with peroxidase as the enzyme. The analysis protocol can be conducted with solution volumes on the order of a few microliters and completed in 40 min. The detection system was capable of detecting 1000 E. coli cells without polymerase chain reaction with high specificity for E. coli vs. the bacteria Bordetella bronchiseptica.     

生物素化ATP硫酸化酶的表达、固定化与应用

现代大规模焦测序技术的产生是DNA测序技术的一次革命,其关键技术之一是得到高活性的、固定于磁性微球表面的ATP硫酸化酶．生物素化的ATP硫酸化酶可以通过生物素与亲和素之间的特异结合特性固定在包被亲和素的磁性微球表面,但是利用化学修饰法将ATP硫酸化酶进行生物素化修饰很可能会影响酶的活性．利用融合表达策略,将大肠杆菌生物素酰基载体蛋白C端87个氨基酸肽段(BCCP87)与ATP硫酸化酶在大肠杆菌内融合表达,经SDS-PAGE和Western blot分析,表达的融合蛋白分子质量约为64 ku,并且能够在大肠杆菌内被生物素化．生物素化的ATP硫酸化酶能够与亲和素包被的磁珠结合,固定后的ATP硫酸化酶具有活性,并且能够用于定量检测焦磷酸盐(PPi)和焦测序,为今后建立高通量大规模焦测序系统提供了一个有效的工具酶．     

Identification and characterization of acetyl-CoA carboxylase gene cluster in <Emphasis Type="Italic">Streptomyces toxytricini</Emphasis>

The gene locus for acetyl-CoA carboxylase (ACC) involved in the primary metabolism was identified from the genomic library of Streptomyces toxytricini which produces a lipase inhibitor lipstatin. The 7.4 kb cloned gene was comprised of 5 ORFs including accD1, accA1, hmgL, fadST1, and stsF. In order to confirm the biochemical characteristics of AccA1, the gene was overexpressed in Escherichia coli cells, and the recombinant protein was purified through Ni²⁺ affinity chromatography. Because most of the expressed AccAl was biotinylated by host E. coli BirA in the presence of D-biotin, the non-biotinylated apo-AccA1 was purified after gene induction without D-biotin, followed by exclusion of holo-AccA1 using streptavidin beads. The separated apo-AccA1 was post-translationally biotinylated by S. toxytricini biotin apo-protein ligase (BPL) in a time- and enzyme-dependent manner. This result supports that this gene cluster of S. toxytricini encodes the functional ACC enzyme subunits to be biotinylated.     

Factors that influence the extracellular expression of streptavidin in <Emphasis Type="Italic">Escherichia coli</Emphasis> using a bacteriocin release protein

Aiming to increase production of recombinant streptavidin in Escherichia coli, the effect of different leader sequences, different promoter strengths of the bacteriocin release protein (kil), host strain and medium composition on the expression and secretion into the medium was investigated. Expression vectors containing an expression or secretion unit were constructed with different combinations of leader sequence for the streptavidin gene and promoters for the kil gene and streptavidin gene. Results showed that a high-level extracellular production of streptavidin could be accomplished with E. coli BL21(DE3) by using the leader sequence of the phoA gene, a strong stationary-phase promoter for the kil gene and supplementation of the medium by glycine. Using a stationary-phase promoter for the expression of streptavidin had a negative effect.     

Immunochemical determination of the cellular content of polypeptide chain release factor RF3 in Escherichia coli

Polypeptide chain termination in Escherchia coli is known to require two codon specific release factors. RF1 and RF2. A third factor, RF3, has been described to stimulate the termination. Earlier investigations have estimated the cellular content of factors RF1 and RF2. Two different immunological techniques for measuring the amount of RF3 per cell in crude E coli cell extracts are reported here, using a sensitive immunoblotting method and a sandwich assay by ELISA. Monoclonal murine antibodies and polyclonal rabbit antibodies were raised against extensively purified recombinant E coli RF3. The immunoblotting involves a specific monoclonal antibody (mAb), biotinylated second antibody and finally radioactive iodinated streptavidin. In the sandwich assay polyclonal antibodies are immobilised on a polystyrene surface before addition of crude cell extract: a specific mAb serves as primary antibody and an HRP-labelled anti-mouse Ig as secondary antibody. Both methods are accurate and rapid to perform. The number of RF3 molecules per cell in exponentially growing E coli cells was found to vary considerably according to the K12 strain examined and depended on the culture medium (from 20 to 500 molecules per cell), faster growth being positively correlated with the number of RF3 molecules per cell.     

Streptavidin-Aequorin Fusion Protein for Bioluminescent Immunoassay

The fusion protein of streptavidin to aequorin (STA-AQ) was highly purified from inclusion bodies in Escherichia coli cells and applied to a bioluminescent sandwich immunoassay. α-Fetoprotein (AFP), which is a serological marker of liver cancer, was used as a model analyte to test STA-AQ in an immunoassay. The measurable range of AFP by the sandwich immunoassay, using the complex of STA-AQ and the biotinylated anti-AFP antibody, was 0.02–200 ng/mL with an average coefficient of variation of 4.9%. The detection sensitivity with the complex of STA-AQ and the biotinylated anti-AFP antibody was similar to that with the complex of biotinylated aequorin, streptavidin and the biotinylated anti-AFP antibody. STA-AQ would be a useful reporter protein for immunoassays.     

Cloning, gene expression and characterization of a novel bacterial NAD-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Neisseria meningitidis strain Z2491

Alignment of the amino acid sequence of some archaeal, bacterial and eukaryotic non-phosphorylating glyceraldehydes-3-phosphate dehydrogenases (GAPNs) and aldehyde dehydrogenases (ALDHs) with the sequence of a putative GAPN present in the genome of the Gram-negative bacterium Neisseria meningitidis strain Z2491 demonstrated the conservation of residues involved in the catalytic activity. The predicted coding sequence of the N. meningitidis gapN gene was cloned in Escherichia coli XL1-blue under the expression of an inducible promoter. The IPTG-induced GAPN was purified ca. 48-fold from E. coli cells using a procedure that sequentially employed conventional ammonium sulfate fractionation as well as anion-exchange and affinity chromatography. The purified recombinant enzyme was thoroughly characterized. The protein is a homotetramer with a 50-kDa subunit, exhibiting absolute specificity for NAD and a broad spectrum of aldehyde substrates. Isoelectric focusing analysis with the purified fraction showed the presence of an acidic polypeptide with an isoelectric point of 6.3. The optimum pH of the purified enzyme was between 9 and 10. Studies on the effect of increasing temperatures on the enzyme activity revealed an optimal value ca. 64 °C. Molecular phylogenetic data suggest that N. meningitidis GAPN has a closer relationship with archaeal GAPNs and glyceraldehyde dehydrogenases than with the typical NADP-specific GAPNs from Gram-positive bacteria and photosynthetic eukaryotes.     

Recombinant Gaussia luciferase with a reactive cysteine residue for chemical conjugation: Expression, purification and its application for bioluminescent immunoassays

The mutated recombinant Gaussia luciferase (hgGLase) having the hinge sequence with a reactive cysteine residue at the carboxyl terminal region was purified from Escherichia coli cells by nickel-chelate affinity chromatography and hydrophobic chromatography. The biotinylated hgGLase (Biotin-hgGLase) was prepared by chemical conjugation with a maleimide activated biotin and apply to bioluminescent immunoassay. In the streptavidin and biotin complex system using Biotin-hgGLase, the measurable range of α-fetoprotein as a model analyte was 0.02–100 ng/ml with the coefficient of variation between 2.5% and 5.2%. The sensitivity of Biotin-hgGLase was similar to that by using the detection system of aequorin, alkaline phosophatase and horseradish peroxidase as a label enzyme.     

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3′ end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A₂₆₆₀, which lies in a sequence that is highly conserved in all species.     

Molecular characterization of polyphosphate kinase (ppk) gene from Serratia marcescens

To understand the mechanism of phosphate accumulation, a gene encoding polyphosphate kinase (PPK) was cloned from the genomic library of Serratia marcescens by Southern hybridization. From the nucleotide sequence of a 4 kb DNA fragment, an open reading frame of 2063 nucleotides was identified encoding a protein of 686 amino acids with molecular mass of 70 kDa. The potential CRP binding site and pho box sequence were found upstream of the putative promoter in the regulatory region. The expression of PPK resulted in the formation of inclusion bodies and the product was active at low temperature. The E. coli strain harboring plasmid pSPK5 with ppk gene increased enzyme activity of polyphosphate kinase, resulting in increased accumulation of polyphosphate in E. coli.     

Accumulation and turnover of 23S ribosomal RNA in azithromycin-inhibited ribonuclease mutant strains of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Ribosomal RNA is normally a stable molecule in bacterial cells with negligible turnover. Antibiotics which impair ribosomal subunit assembly promote the accumulation of subunit intermediates in cells which are then degraded by ribonucleases. It is predicted that cells expressing one or more mutated ribonucleases will degrade the antibiotic-bound particle less efficiently, resulting in increased sensitivity to the antibiotic. To test this, eight ribonuclease-deficient strains of Escherichia coli were grown in the presence or absence of azithromycin. Cell viability and protein synthesis rates were decreased in these strains compared with wild type cells. Degradation of 23S rRNA and recovery from azithromycin inhibition were examined by ³H-uridine labeling and by hybridization with a 23S rRNA specific probe. Mutants defective in ribonuclease II and polynucleotide phosphorylase demonstrated hypersensitivity to the antibiotic and showed a greater extent of 23S rRNA accumulation and a slower recovery rate. The results suggest that these two ribonucleases are important in 23S rRNA turnover in antibiotic-inhibited E. coli cells.     

Development of a Streptavidin-Conjugated Single-Chain Antibody That Binds Bacillus cereus Spores

Control of microorganisms such as Bacillus cereus spores is critical to ensure the safety and a long shelf life of foods. A bifunctional single chain antibody has been developed for detection and binding of B. cereus T spores. The genes that encode B. cereus T spore single-chain antibody and streptavidin were connected for use in immunoassays and immobilization of the recombinant antibodies. A truncated streptavidin, which is smaller than but has biotin binding ability similar to that of streptavidin, was used as the affinity domain because of its high and specific affinity with biotin. The fusion protein gene was expressed in Escherichia coli BL21 (DE3) with the T7 RNA polymerase-T7 promoter expression system. Immunoblotting revealed an antigen specificity similar to that of its parent native monoclonal antibody. The single-chain antibody-streptavidin fusion protein can be used in an immunoassay of B. cereus spores by applying a biotinylated enzyme detection system. The recombinant antibodies were immobilized on biotinylated magnetic beads by taking advantage of the strong biotin-streptavidin affinity. Various liquids were artificially contaminated with 5 × 10⁴ B. cereus spores per ml. Greater than 90% of the B. cereus spores in phosphate buffer or 37% of the spores in whole milk were tightly bound and removed from the liquid phase by the immunomagnetic beads.     

Rapid Detection, Identification, and Enumeration of Escherichia coli Cells in Municipal Water by Chemiluminescent In Situ Hybridization

A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturable Escherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35°C, individual microcolonies of E. coli were detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targeting P. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other than E. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.     

Expression of the Neisseria gonorrhoeae major outer membrane protein PI in Escherichia coli

Summary To actively express an outer membrane protein, protein I (PI), from different strains of Neisseria gonorrhoeae in E.␣coli, PI gene fragments from two reference strains and four clinical isolates of Neisseria gonorrhoeae were obtained with PCR amplification. They were cloned into the PCR cloning vector pBS-T to form pBS-T-PI and sequenced. Subsequently, they were cloned into an expression vector pET-30b (+) to generate pET-PI recombinants. After inducing with isopropyl-β-d-thiogalactopyranoside (IPTG), the expressed PI proteins were analysed by SDS-PAGE, Western blotting and ELISA. The results implied that we had successfully constructed the PI gene recombinants from both reference strains and clinical isolates and obtained the recombinant proteins expressed in E. coli at relatively high levels, and the expressed proteins had the immunological activity with the corresponding antibodies. This research will be very helpful for the further study of these proteins in generating preventive vaccines on Neisseria gonorrhoeae infection and clinical diagnosis.     

Expression study with the Escherichia coli lep gene for leader peptidase in phototrophic purple bacteria

Synthesis and assembly of leader peptidase of Escherichia coli (signal peptidase I), was studied by heterologous expression of its lep gene in three species of phototrophic purple bacteria. Cell extracts of the recipient species showed neither cross reaction with antibodies against E. coli leader peptidase nor cleavage of the model substrate M13-procoat in vitro. The lep gene was transferred via conjugation using the plasmid expression vector for phototrophic bacteria pJAJ9. Plasmidborne leader peptidase enzyme was identified by immunochemical means. However, extracts of transconjugant cells showed no cleavage function. Trypsin digestion studies revealed that the enzyme was not properly integrated across the host membranes. The data suggest that cleaving enzymes for protein export and/or their assembly pathway in purple bacteria differ from the E. coli type.Abbreviations DMSO dimethylsulfoxide - EDTA ethylenediamine tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate     

Binding of the S7 Protein with Fragment 926–986/1219–1393 of the 16S rRNA As a Key Step in the Assembly of the Small Subunit of Prokaryotic Ribosomes

Both structural and thermodynamic studies are necessary to understand the ribosome assembly. An initial step was made in studying the interaction between a 16S rRNA fragment and S7, a key protein in assembling the prokaryotic ribosome small subunit. The apparent dissociation constant was obtained for complexes of recombinant Escherichia coliandThermus thermophilusS7 with a fragment of the 3" domain of the E. coli16S rRNA. Both proteins showed high rRNA-binding activity, which was not observed earlier. Since RNA and proteins are conformationally labile, their folding must be considered to correctly describe the RNA–protein interactions.     

鲑鱼生长激素基因分泌型表达质粒的构建

生长激素(GH)是动物垂体前叶分泌的一种多肽类激素.应用分子重组及PCR等技术,构建了一种鲑鱼生长激素基因分泌型表达质粒pOsGH153,使编码鲑鱼生长激素成熟肽的序列克隆在大肠杆菌分泌型表达载体PIN-Ⅲ-ompA内,直接位于编码大肠杆菌外膜蛋白A信号肽序列的下游,在Lpp-Lac杂合启动子控制下,经IPTG诱导,分子量约23 000的鲑鱼生长激素在大肠杆菌中获得高效表达,该产物具有天然鲑鱼生长激素的免疫活性,直接分泌到细胞周质,而信号肽被自动剪除.     

Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance

A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5′-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104 mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5′-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.     

Identification of an Escherichia coli S1-like protein in the spinach chloroplast ribosome

Antibodies directed against E. coli ribosomal protein S1 were used in immunoblotting assays to search for an S1-like protein in the ribosome of spinach chloroplast. An immunological cross-reaction was reproducibly detected on the blots and inhibition experiments have demonstrated its specificity. The chloroplastic ribosomal protein which has epitopes common to antigenic determinants of the E. coli protein S1 was identified as being protein S2/S3.     

Characterization of a genomic clone of Erwinia carotovora subsp. carotovora TG3 encoding a pectolytic enzyme of apparent molecular weight 78 kDa

Summary A clone containing the gene encoding a pectolytic enzyme of Erwinia carotovora subsp. carotovora was selected as the one that showed maceration on a solid medium containing sodium polypectate. The gene was located on a 3.2-kb DNA fragment flanked by a BglII site and a Hin-dIII site. Via mini-Mudlac mutagenesis, a possible promoter site was located within the gene between the BglII site and the EcoRI site. The mRNA transcribed from the promoter was directed from the BglII site toward the EcoRI site, determined from the orientation of the inserted mini-Mudlac. The probable gene product was identified as a 78 kDa protein. The enzyme activity of the Escherichia coli clone was detected mainly in the periplasmic space. Potato tuber slices were not macerated by the E. coli clone and synthesis of the enzyme in E. coli was not regulated by the enzyme substrate, sodium polypectate.