scholarly journals I-Block: a simple Escherichia coli-based assay for studying sequence-specific DNA binding of proteins

2020 ◽  
Vol 48 (5) ◽  
pp. e28-e28
Author(s):  
Sarolta Szentes ◽  
Nikolett Zsibrita ◽  
Mihály Koncz ◽  
Eszter Zsigmond ◽  
Pál Salamon ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Specific Binding ◽  
Lac Repressor ◽  
Host Bacterium ◽  
Lambda Phage ◽  
Simple Method ◽  
E Coli ◽  
Laci Gene ◽  
Compatible Plasmid ◽  
Nhei Site

Abstract We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive β-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased β-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.

scholarly journals Lectin and E. coli Binding to Carbohydrate-Functionalized Oligo(ethylene glycol)-Based Microgels: Effect of Elastic Modulus, Crosslinker and Carbohydrate Density

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 263
Author(s):  
Fabian Schröer ◽  
Tanja J. Paul ◽  
Dimitri Wilms ◽  
Torben H. Saatkamp ◽  
Nicholas Jäck ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Elastic Modulus ◽  
Ethylene Glycol ◽  
Concanavalin A ◽  
Specific Binding ◽  
Network Density ◽  
Carbohydrate Binding ◽  
E Coli ◽  
Surface Recognition ◽  
Strong Anchoring

The synthesis of carbohydrate-functionalized biocompatible poly(oligo(ethylene glycol) methacrylate microgels and the analysis of the specific binding to concanavalin A (ConA) and Escherichia coli (E. coli) is shown. By using different crosslinkers, the microgels’ size, density and elastic modulus were varied. Given similar mannose (Man) functionalization degrees, the softer microgels show increased ConA uptake, possibly due to increased ConA diffusion in the less dense microgel network. Furthermore, although the microgels did not form clusters with E. coli in solution, surfaces coated with mannose-functionalized microgels are shown to bind the bacteria whereas galactose (Gal) and unfunctionalized microgels show no binding. While ConA binding depends on the overall microgels’ density and Man functionalization degree, E. coli binding to microgels’ surfaces appears to be largely unresponsive to changes of these parameters, indicating a rather promiscuous surface recognition and sufficiently strong anchoring to few surface-exposed Man units. Overall, these results indicate that carbohydrate-functionalized biocompatible oligo(ethylene glycol)-based microgels are able to immobilize carbohydrate binding pathogens specifically and that the binding of free lectins can be controlled by the network density.

scholarly journals Characterization of the β-lactamase specified by the resistance factor R-1818 in Escherichia coli K12 and other Gram-negative bacteria

1971 ◽  
Vol 123 (4) ◽  
pp. 501-505 ◽  
Author(s):  
J. W. Dale
Keyword(s):  
Escherichia Coli ◽  
Host Species ◽  
Host Bacterium ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
R Factor ◽  
Relationship Of ◽  
The Relationship

1. The amino acid composition of the β-lactamase from E. coli (R-1818) was determined. 2. The R-1818 β-lactamase is inhibited by formaldehyde, hydroxylamine, sodium azide, iodoacetamide, iodine and sodium chloride. 3. The Km values for benzylpenicillin, ampicillin and oxacillin have been determined by using the R-factor enzyme from different host species. The same values were obtained, irrespective of the host bacterium. 4. The molecular weight of the enzyme was found to be 44600, and was the same for all host species. 5. The relationship of R-1818 and R-GN238 β-lactamases is discussed.

Evaluation of biofilm performance as a protective barrier against biocorrosion using an enzyme electrode

2011 ◽  
Vol 64 (8) ◽  
pp. 1736-1742 ◽  
Author(s):  
S. Soleimani ◽  
B. Ormeci ◽  
O. B. Isgor ◽  
S. Papavinasam
Keyword(s):  
Escherichia Coli ◽  
Growth Conditions ◽  
Microbiologically Influenced Corrosion ◽  
Simple Method ◽  
E Coli ◽  
Concrete Deterioration ◽  
Protective Barrier ◽  
Microbial Biofilms ◽  
Short Time ◽  
Selection Of

Sulfide is known to be an important factor in microbiologically influenced corrosion (MIC) of metals and concrete deterioration in wastewater treatment structures and sewer pipelines. A sulfide biosensor was used to determine the effectiveness of Escherichia coli DH5α biofilm as a protective barrier against MIC. The biofilm was shown to be effective in protecting surfaces from sulfide and helping to reduce MIC using amperometric measurements. The results also indicated that the growth conditions of E. coli DH5α may have an impact on the performance of the biofilm as a sulfide barrier. The simple method provided in this work enables the comparison of several microbial biofilms and selection of the ones with potential to prevent MIC in a relatively short time.

scholarly journals Anti-Escherichia coli Functionalized Silver-Doped Carbon Nanofibers for Capture of E. coli in Microfluidic Systems

Polymers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1117 ◽  
Author(s):  
Soshana Smith ◽  
Michael Delaney ◽  
Margaret Frey
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Carbon Nanofibers ◽  
Specific Binding ◽  
Base Material ◽  
Fiber Surface ◽  
Electrospinning Process ◽  
Silver Particles ◽  
Microfluidic Systems ◽  
E Coli

Silver-doped carbon nanofibers (SDCNF) are used as the base material for the selective capture of Escherichia coli in microfluidic systems. Fibers were spun in a glovebox with dry atmosphere maintained by forced dry air pumped through the closed environment. This affected the evaporation rate of the solvent during the electrospinning process and the distribution of silver particles within the fiber. Antibodies are immobilized on the surface of the silver-doped polyacrylonitrile (PAN) based carbon nanofibers via a three-step process. The negatively charged silver particles present on the surface of the nanofibers provide suitable sites for positively charged biotinylated poly-(L)-lysine-graft-poly-ethylene-glycol (PLL-g-PEG biotin) conjugate attachment. Streptavidin and a biotinylated anti-E. coli antibody were then added to create anti-E. coli surface functionalized (AESF) nanofibers. Functionalized fibers were able to immobilize up to 130 times the amount of E. coli on the fiber surface compared to neat silver doped fibers. Confocal images show E. coli remains immobilized on fiber mat surface after extensive rinsing showing the bacteria is not simply a result of non-specific binding. To demonstrate selectivity and functionalization with both gram negative and gram-positive antibodies, anti-Staphylococcus aureus surface functionalized (ASSF) nanofibers were also prepared. Experiments with AESF performed with Staphylococcus aureus (S. aureus) and ASSF with E. coli show negligible binding to the fiber surface showing the selectivity of the functionalized membranes. This surface functionalization can be done with a variety of antibodies for tunable selective pathogen capture.

scholarly journals Adaptation in a Mouse Colony Monoassociated with Escherichia coli K-12 for More than 1,000 Days

2010 ◽  
Vol 76 (14) ◽  
pp. 4655-4663 ◽  
Author(s):  
Sean M. Lee ◽  
Aaron Wyse ◽  
Aaron Lesher ◽  
Mary Lou Everett ◽  
Linda Lou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rates ◽  
Bacterial Species ◽  
Intestinal Flora ◽  
Average Density ◽  
Host Bacterium ◽  
E Coli ◽  
K 12

ABSTRACT Although mice associated with a single bacterial species have been used to provide a simple model for analysis of host-bacteria relationships, bacteria have been shown to display adaptability when grown in a variety of novel environments. In this study, changes associated with the host-bacterium relationship in mice monoassociated with Escherichia coli K-12 over a period of 1,031 days were evaluated. After 80 days, phenotypic diversification of E. coli was observed, with the colonizing bacteria having a broader distribution of growth rates in the laboratory than the parent E. coli. After 1,031 days, which included three generations of mice and an estimated 20,000 generations of E. coli, the initially homogeneous bacteria colonizing the mice had evolved to have widely different growth rates on agar, a potential decrease in tendency for spontaneous lysis in vivo, and an increased tendency for spontaneous lysis in vitro. Importantly, mice at the end of the experiment were colonized at an average density of bacteria that was more than 3-fold greater than mice colonized on day 80. Evaluation of selected isolates on day 1,031 revealed unique restriction endonuclease patterns and differences between isolates in expression of more than 10% of the proteins identified by two-dimensional electrophoresis, suggesting complex changes underlying the evolution of diversity during the experiment. These results suggest that monoassociated mice might be used as a tool for characterizing niches occupied by the intestinal flora and potentially as a method of targeting the evolution of bacteria for applications in biotechnology.

scholarly journals Specific Secretion of Active Single-Chain Fv Antibodies into the Supernatants of Escherichia coliCultures by Use of the Hemolysin System

2000 ◽  
Vol 66 (11) ◽  
pp. 5024-5029 ◽  
Author(s):  
Luis A. Fernández ◽  
Isabel Sola ◽  
Luis Enjuanes ◽  
Víctor de Lorenzo
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Culture Media ◽  
Single Chain ◽  
Extracellular Medium ◽  
Simple Method ◽  
E Coli ◽  
Single Chain Fv ◽  
Bacterial Cytoplasm ◽  
Culture Supernatants

ABSTRACT A simple method for the nontoxic, specific, and efficient secretion of active single-chain Fv antibodies (scFvs) into the supernatants ofEscherichia coli cultures is reported. The method is based on the well-characterized hemolysin transport system (Hly) of E. coli that specifically secretes the target protein from the bacterial cytoplasm into the extracellular medium without a periplasmic intermediate. The culture media that accumulate these Hly-secreted scFv's can be used in a variety of immunoassays without purification. In addition, these culture supernatants are stable over long periods of time and can be handled basically as immune sera.

Recirculating Immunomagnetic Separation and Optimal Enrichment Conditions for Enhanced Detection and Recovery of Low Levels of Escherichia coli O157:H7 from Fresh Leafy Produce and Surface Water

2007 ◽  
Vol 70 (12) ◽  
pp. 2717-2724 ◽  
Author(s):  
SUNEE HIMATHONGKHAM ◽  
MARY LEE DODD ◽  
JENNY K. YEE ◽  
DAVID K. LAU ◽  
RAYMOND G. BRYANT ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Surface Water ◽  
Real Time ◽  
Real Time Pcr ◽  
Immunomagnetic Separation ◽  
Escherichia Coli O157 ◽  
Simple Method ◽  
E Coli ◽  
Low Levels ◽  
Spinach Leaves

The objective of this study was to develop a rapid, simple method for enhanced detection and isolation of low levels of Escherichia coli O157:H7 from leafy produce and surface water using recirculating immunomagnetic separation (RIMS) coupled with real-time PCR and a standard culture method. The optimal enrichment conditions for the method also were determined. Analysis of real-time PCR data (CT values) suggested that incubation of lettuce and spinach leaves rather than rinsates provides better enrichment of E. coli O157:H7. Enrichment of lettuce or spinach leaves at 42°C for 5 h provided better detection than enrichment at 37°C. Extended incubation of surface water for 20 h at 42°C did not improve the detection. The optimized enrichment conditions were also employed with modified Moore swabs, which were used to sample flowing water sites. Positive isolation rates and real-time PCR results indicated an increased recovery of E. coli O157:H7 from all samples following the application of RIMS. Under these conditions, the method provided detection and/or isolation of E. coli O157:H7 at levels as low as 0.07 CFU/g of lettuce, 0.1 CFU/g of spinach, 6 CFU/100 ml of surface water, and 9 CFU per modified Moore swab. During a 6-month field study, modified Moore swabs yielded high isolation rates when deployed in natural watershed sites. The method used in this study was effective for monitoring E. coli O157:H7 in the farm environment, during postharvest processing, and in foodborne outbreak investigations.

scholarly journals Molecular Characterization of the EhaG and UpaG Trimeric Autotransporter Proteins from Pathogenic Escherichia coli

2012 ◽  
Vol 78 (7) ◽  
pp. 2179-2189 ◽  
Author(s):  
Makrina Totsika ◽  
Timothy J. Wells ◽  
Christophe Beloin ◽  
Jaione Valle ◽  
Luke P. Allsopp ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Specific Binding ◽  
Negative Regulator ◽  
Passenger Domain ◽  
Content Type ◽  
E Coli ◽  
Ecm Proteins

ABSTRACTTrimeric autotransporter proteins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. A common feature of most TAAs is the ability to mediate adherence to eukaryotic cells or extracellular matrix (ECM) proteins via a cell surface-exposed passenger domain. Here we describe the characterization of EhaG, a TAA identified from enterohemorrhagicEscherichia coli(EHEC) O157:H7. EhaG is a positional orthologue of the recently characterized UpaG TAA from uropathogenicE. coli(UPEC). Similarly to UpaG, EhaG localized at the bacterial cell surface and promoted cell aggregation, biofilm formation, and adherence to a range of ECM proteins. However, the two orthologues display differential cellular binding: EhaG mediates specific adhesion to colorectal epithelial cells while UpaG promotes specific binding to bladder epithelial cells. The EhaG and UpaG TAAs contain extensive sequence divergence in their respective passenger domains that could account for these differences. Indeed, sequence analyses of UpaG and EhaG homologues from severalE. coligenomes revealed grouping of the proteins in clades almost exclusively represented by distinctE. colipathotypes. The expression of EhaG (in EHEC) and UpaG (in UPEC) was also investigated and shown to be significantly enhanced in anhnsisogenic mutant, suggesting that H-NS acts as a negative regulator of both TAAs. Thus, while the EhaG and UpaG TAAs contain some conserved binding and regulatory features, they also possess important differences that correlate with the distinct pathogenic lifestyles of EHEC and UPEC.

scholarly journals Characterization of a mammalian homolog of the Escherichia coli MutY mismatch repair protein.

1995 ◽  
Vol 15 (2) ◽  
pp. 989-996 ◽  
Author(s):  
J P McGoldrick ◽  
Y C Yeh ◽  
M Solomon ◽  
J M Essigmann ◽  
A L Lu
Keyword(s):  
Escherichia Coli ◽  
Specific Binding ◽  
Polyclonal Antibodies ◽  
Gradient Centrifugation ◽  
Immunoblot Analysis ◽  
Partial Purification ◽  
Phosphodiester Bond ◽  
E Coli ◽  
Nuclear Extracts ◽  
Calf Thymus

A protein homologous to the Escherichia coli MutY protein, referred to as MYH, has been identified in nuclear extracts of calf thymus and human HeLa cells. Western blot (immunoblot) analysis using polyclonal antibodies to the E. coli MutY protein detected a protein of 65 kDa in both extracts. Partial purification of MYH from calf thymus cells revealed a 65-kDa protein as well as a functional but apparently degraded form of 36 kDa, as determined by glycerol gradient centrifugation and immunoblotting with anti-MutY antibodies. Calf MYH is a DNA glycosylase that specifically removes mispaired adenines from A/G, A/7,8-dihydro-8-oxodeoxyguanine (8-oxoG or GO), and A/C mismatches (mismatches indicated by slashes). A nicking activity that is either associated with or copurified with MYH was also detected. Nicking was observed at the first phosphodiester bond 3' to the apurinic or apyrimidinic (AP) site generated by the glycosylase activity. The nicking activity on A/C mismatches was 30-fold lower and the activity on A/GO mismatches was twofold lower than that on A/G mismatches. No nicking activity was detected on substrates containing other selected mismatches or homoduplexes. Nicking activity on DNA containing A/G mismatches was inhibited in the presence of anti-MutY antibodies or upon treatment with potassium ferricyanide, which oxidizes iron-sulfur clusters. Gel shift analysis showed specific binding complex formation with A/G and A/GO substrates, but not with A/A, C.GO, and C.G substrates. Binding is sevenfold greater on A/GO substrates than on A/G substrates. The eukaryotic MYH may be involved in the major repair of both replication errors and oxidative damage to DNA, the same functions as those of the E. coli MutY protein.

scholarly journals Topical Application of Escherichia coli-Vectored Vaccine as a Simple Method for Eliciting Protective Immunity

2006 ◽  
Vol 74 (6) ◽  
pp. 3607-3617 ◽  
Author(s):  
Jianfeng Zhang ◽  
Zhongkai Shi ◽  
Fan-kun Kong ◽  
Edward Jex ◽  
Zhigang Huang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Topical Application ◽  
Defense Mechanism ◽  
Clostridium Tetani ◽  
Simple Method ◽  
Γδt Cells ◽  
E Coli ◽  
Innate Defense ◽  
Γδt Cell ◽  
Vectored Vaccine

ABSTRACT We report here that animals can be protected against lethal infection by Clostridium tetani cells and Bacillus anthracis spores following topical application of intact particles of live or γ-irradiated Escherichia coli vectors overproducing tetanus and anthrax antigens, respectively. Cutaneous γδT cells were rapidly recruited to the administration site. Live E. coli cells were not found in nonskin tissues after topical application, although fragments of E. coli DNA were disseminated transiently. Evidence suggested that intact E. coli particles in the outer layer of skin may be disrupted by a γδT-cell-mediated innate defense mechanism, followed by the presentation of E. coli ligand-adjuvanted intravector antigens to the immune system and rapid degradation of E. coli components. The nonreplicating E. coli vector overproducing an exogenous immunogen may foster the development of a new generation of vaccines that can be manufactured rapidly and administered noninvasively in a wide variety of disease settings.

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