Latex Test for Quantitative Determination of Escherichia coli Antibody

A latex agglutination test was developed for assay of anti-Escherichia coli antisera. The test is simple, specific, sensitive, and reproducible.     

抗噬菌体工程菌的筛选

噬菌体污染常引起溶菌,造成人力物力浪费。应用自发突变的原理成功地从溶菌液中筛选到抗噬菌体的工程菌株;在发酵罐中培养,该菌株生长行为和表达水平没有变化。     

Induction of Lambda-Bacteriophage in Escherichia coli as a Screening Test for Potential Antitumor Agents

A simple, rapid, quantitative test procedure to measure induction of phage production in lysogenic Escherichia coli K-12 (λ) was described. This test was used in a study of 209 substances, including antibiotics, pyrimirines, purines, alkylating agents, thiols, amino acids, vitamins, and miscellaneous compounds. Minimal inducing concentrations for the 26 (12.5% of total tested) substances found to be effective inducing agents, as well as a listing of the inactive compounds, are presented. Since 21 of the 26 active agents reportedly have antineoplastic activity in rodent tumor systems, it was concluded that the induction test may provide a useful screen for the detection of potentially useful antitumor compounds.     

Screening Procedure for Escherichia coli Mutants Deficient in Ribonuclease I

Strains of Escherichia coli that possess ribonucleic acid accumulated under relaxed growth conditions show a considerable increase in time before the onset of beta-galactosidase inducibility. This time dependency can be related to the presence or absence of ribonuclease I.     

Screening of genetic parameters for soluble protein expression in Escherichia coli

Soluble expression of proteins in a relevant form for functional and structural investigations still often remains a challenge. Although many biochemical factors are known to affect solubility, a thorough investigation of yield-limiting factors is normally not feasible in high-throughput efforts. Here we present a screening strategy for expression of biomedically relevant proteins in Escherichia coli using a panel of six different genetic variations. These include engineered strains for rare codon supplementation, increased disulfide bond formation in the cytoplasm and novel vectors for secretion to the periplasm or culture medium. Combining these variants with expression construct truncations design, we report on parallel cloning and expression of more than 300 constructs representing 24 selected proteins; including full-length variants of human growth factors, interleukins and growth factor binding proteins. This rapid screening approach appears highly suitable for high-throughput efforts targeting either large sets of proteins or more focused investigations regarding individual high-profile targets.     

Serum and Egg Antibody Responses in Chickens to Escherichia coli

The effects of Freund’s adjuvants on antibody production in chickens against E. coli whole cells were examined. The levels of anti-E. coli IgG antibodies in serum were higher when Freund’s complete (FCA) or incomplete adjuvant (FIA) was administered than that without adjuvant. Production of antibodies recognizing E. coli cells and their lipopolysaccharide was enhanced by FIA, while both FIA and FCA enhanced production of antibodies recognizing outer membrane components. In contrast, serum IgM antibody levels were higher when no adjuvant was used. Anti-E. coli IgG antibodies in serum were efficiently transferred to egg yolk, giving antibody activity in egg yolk similar to that in serum. However, anti-E. coli IgM antibodies were not detected in the egg, suggesting that egg (white) IgM was not influenced by antigenic stimulation of the humoral immune system. Antimicrobial activity of the egg yolk IgG was highest when the bacteria antigen was injected with FIA.     

抗镰刀菌单链抗体在大肠杆菌中可溶性表达条件的研究

通过优化诱导表达条件,实现抗镰刀菌单链抗体在大肠杆菌周质中高效可溶性表达。将抗镰刀菌单链抗体FvSG7转化到大肠杆菌XL1-Blue中,确定诱导表达培养基,在不同的诱导温度、诱导剂IPTG的浓度和诱导时间条件下培养重组大肠杆菌,通过Western杂交和ELISA检测分析单链抗体的可溶性表达情况以及抗体的活性。重组大肠杆菌培养至OD600nm为0.5时加入终浓度为0.1 mmol/L IPTG,25℃诱导表达2 h可获得最大量的可溶性单链抗体。通过对诱导温度、IPTG浓度和诱导时间等表达条件的优化,可以显著提高Fv SG7抗体在大肠杆菌XL1-Blue周质中的表达量。     

抗甜菜坏死黄脉病毒单链抗体表达载体的构建及其表达

用ＰＣＲ方法以分泌抗甜菜坏死黄脉病毒（ＢＮＹＶＶ）单克隆抗体的杂交瘤细胞的基因组为模板,扩增了编码ＢＮＹＶＶ单抗的重链可变区（ＶＨ）基因。测序表明,该ＶＨ序列属于小鼠ＩＩ（Ａ）亚类,全长为３６０ｂｐ,编码１２０个氨基酸。将其和先前克隆的轻链基因分别插入到一个含有连接ＶＨ和ＶＬ基因的连接序列的质粒之中,构建成单链抗体（ｓｃＦｖ）基因的表达载体ｐＴＣｓｃＦｖ。将质粒在大肠杆菌中表达,ＥＬＩＳＡ法检测出     

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and β-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols¹. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production². Of particular interest are fungal cellulases^3-8, which are already being used industrially for foods and textiles processing.Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina^3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina¹⁰ and Phanerochaete chrysosporium^11-12. We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (“halos”) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. Traditional bacterial whole cell CMC/CR activity assays¹³ involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods¹⁴ for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.Download video file.^{(38M, mov)}     

Comparison of Fluorescent Antibody with Cultural Technique for Isolation of Enteropathogenic Escherichia coli from Swine

In an investigation of hogs as possible reservoirs of human strains of enteropathogenic Escherichia coli (EEC), 92 six-month-old grain- and garbage-fed hogs were examined on the farm and again at the packing plant. Of the 331 specimens obtained by swabbing the rectum, cecum, and edible meat carcass of these hogs, 125 were presumptively positive for EEC when screened by the fluorescent-antibody (FA) technique. These “presumptive positive” specimens then underwent extensive bacteriological examination and complete serological typing. The FA technique proved to be an easier, simpler, and more economical procedure than culture when a large number of specimens were examined for possible EEC serogroups. It was found especially valuable for identification of multiple serogroups of EEC within a single specimen. It also appeared to be more sensitive than cultural examination, since results were not dependent on the presence of large numbers of organisms in the specimen, or even on their viability. However, the FA technique was found to be less specific than culture because of cross-reactivity with antigenically related Enterobacteriaceae when fluorescein-labeled antisera were used. Therefore, any specimen found positive on FA examination should be considered as presumptive positive until confirmed by bacteriological examination and complete serological study.     

Development of a Rapid Agglutination Latex Test for Diagnosis of Enteropathogenic and Enterohemorrhagic Escherichia coli Infection in Developing World: Defining the Biomarker,Antibody and Method

Background

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC/EHEC) are human intestinal pathogens responsible for diarrhea in both developing and industrialized countries. In research laboratories, EPEC and EHEC are defined on the basis of their pathogenic features; nevertheless, their identification in routine laboratories is expensive and laborious. Therefore, the aim of the present work was to develop a rapid and simple assay for EPEC/EHEC detection. Accordingly, the EPEC/EHEC-secreted proteins EspA and EspB were chosen as target antigens.

Methodology

First, we investigated the ideal conditions for EspA/EspB production/secretion by ELISA in a collection of EPEC/EHEC strains after cultivating bacterial isolates in Dulbecco’s modified Eagle’s medium (DMEM) or DMEM containing 1% tryptone or HEp-2 cells-preconditioned DMEM, employing either anti-EspA/anti-EspB polyclonal or monoclonal antibodies developed and characterized herein. Subsequently, a rapid agglutination latex test (RALT) was developed and tested with the same collection of bacterial isolates.

Principal findings

EspB was defined as a biomarker and its corresponding monoclonal antibody as the tool for EPEC/EHEC diagnosis; the production of EspB was better in DMEM medium. RALT assay has the sensitivity and specificity required for high-impact diagnosis of neglected diseases in the developing world.

Conclusion

RALT assay described herein can be considered an alternative assay for diarrhea diagnosis in low-income countries since it achieved 97% sensitivity, 98% specificity and 97% efficiency.     

A pathway for putrescine catabolism in Escherichia coli

Escherichia coli mutants able to grow in putrescine have been isolated from gamma-aminobutyrate mutants. These mutants show putrescine-alpha-ketoglutarate transaminase and gamma-aminobutyraldehyde dehydrogenase activities. Both enzymes have been characterized, the first of them showing an apparent Km for putrescine of 22.5 microM and the second an apparent Km of 37 microM for NAD and 18 microM for delta-1-pyrroline; the optimum pH values were 7.2 and 5.4, respectively, for the two enzymes.     

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.     

A PCR-ELISA for detecting Shiga toxin-producing Escherichia coli

A sensitive and specific PCR-ELISA was developed to detect Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) in food. The assay was based on the incorporation of digoxigenin-labeled dUTP and a biotin-labeled primer specific for Shiga toxin genes during PCR amplification. The labeled PCR products were bound to streptavidin-coated wells of a microtiter plate and detected by an ELISA. The specificity of the PCR was determined using 39 bacterial strains, including STEC, enteropathogenic E. coli, E. coli K12, and Salmonella. All of the STEC strains were positive, and non-STEC organisms were negative. The ELISA detecting system was able to increase the sensitivity of the PCR assay by up to 100-fold, compared with a conventional gel electrophoresis. The detection limit of the PCR-ELISA was 0.1-10 CFU dependent upon STEC serotypes, and genotypes of Shiga toxins. With the aid of a simple DNA extraction system, PrepMan, the PCR-ELISA was able to detect ca. 10(5) CFU of STEC per gram of ground beef without any culture enrichment. The entire procedure took about 6 h. Because of its microtiter plate format, PCR-ELISA is particularly suitable for large-scale screening and compatible with future automation.