One-Step Preparation of Competent E. coli: Transformation and Storage of Bacterial Cells in the Same Solution

2021 ◽  
Vol 2021 (11) ◽  
pp. pdb.prot101212 ◽  
Author(s):  
Michael R. Green ◽  
Joseph Sambrook
Keyword(s):  
Escherichia Coli ◽  
Convenient Method ◽  
Plasmid Dna ◽  
Transformation Efficiency ◽  
Bacterial Cells ◽  
E Coli ◽  
One Step ◽  
And Storage

This protocol describes a convenient method for the preparation, use, and storage of competent Escherichia coli. The reported transformation efficiency of this method is ∼5 × 107 transformants/µg of plasmid DNA.

PBP4 and PBP5 are involved in regulating exopolysaccharide synthesis during Escherichia coli biofilm formation

Microbiology ◽  
2021 ◽  
Vol 167 (3) ◽  
Author(s):  
Sathi Mallick ◽  
Shanti Kiran ◽  
Tapas Kumar Maiti ◽  
Anindya S. Ghosh
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Type Species ◽  
Pentose Phosphate ◽  
Bacterial Cells ◽  
Surface Adhesion ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Link Type

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

scholarly journals Bacterial Secretions of Nonpathogenic Escherichia coli Elicit Inflammatory Pathways: a Closer Investigation of Interkingdom Signaling

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Amin Zargar ◽  
David N. Quan ◽  
Karen K. Carter ◽  
Min Guo ◽  
Herman O. Sintim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Negative Feedback ◽  
Cell Contact ◽  
Bacterial Cells ◽  
Content Type ◽  
Phenotypic Differences ◽  
E Coli ◽  
Autoinducer 2 ◽  
Human Epithelial Cells

ABSTRACTThere have been many studies on the relationship between nonpathogenic bacteria and human epithelial cells; however, the bidirectional effects of the secretomes (secreted substances in which there is no direct bacterium-cell contact) have yet to be fully investigated. In this study, we use a transwell model to explore the transcriptomic effects of bacterial secretions from two different nonpathogenicEscherichia colistrains on the human colonic cell line HCT-8 using next-generation transcriptome sequencing (RNA-Seq).E. coliBL21 and W3110, while genetically very similar (99.1% homology), exhibit key phenotypic differences, including differences in their production of macromolecular structures (e.g., flagella and lipopolysaccharide) and in their secretion of metabolic byproducts (e.g., acetate) and signaling molecules (e.g., quorum-sensing autoinducer 2 [AI-2]). After analysis of differential epithelial responses to the respective secretomes, this study shows for the first time that a nonpathogenic bacterial secretome activates the NF-κB-mediated cytokine-cytokine receptor pathways while also upregulating negative-feedback components, including the NOD-like signaling pathway. Because of AI-2's relevance as a bacterium-bacterium signaling molecule and the differences in its secretion rates between these strains, we investigated its role in HCT-8 cells. We found that the expression of the inflammatory cytokine interleukin 8 (IL-8) responded to AI-2 with a pattern of rapid upregulation before subsequent downregulation after 24 h. Collectively, these data demonstrate that secreted products from nonpathogenic bacteria stimulate the transcription of immune-related biological pathways, followed by the upregulation of negative-feedback elements that may serve to temper the inflammatory response.IMPORTANCEThe symbiotic relationship between the microbiome and the host is important in the maintenance of human health. There is a growing need to further understand the nature of these relationships to aid in the development of homeostatic probiotics and also in the design of novel antimicrobial therapeutics. To our knowledge, this is the first global-transcriptome study of bacteria cocultured with human epithelial cells in a model to determine the transcriptional effects of epithelial cells in which epithelial and bacterial cells are allowed to “communicate” with each other only through diffusible small molecules and proteins. By beginning to demarcate the direct and indirect effects of bacteria on the gastrointestinal (GI) tract, two-way interkingdom communication can potentially be mediated between host and microbe.

scholarly journals Effect of Gold Nanoparticles Size Capped with Surfactant on the Transformation of Plasmid into Escherichia coli Bacteria

2021 ◽  
Vol 5 (1) ◽  
Author(s):  
Amr D ◽  
◽  
Attia N ◽  
Seufi A ◽  
Galal A ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gold Nanoparticles ◽  
Heat Shock ◽  
Transformation Efficiency ◽  
Transformation Process ◽  
Bacterial Cells ◽  
Bacterial Transformation ◽  
Foreign Genes ◽  
Escherichia Coli Bacteria ◽  
Convenient Technique

Bacterial transformation has great importance in molecular biology, as it is used for introduction of foreign genes into bacterial cells either chemical or physical ways. Using calcium chloride to prepare competent cells and heat shock is the most widely used method for bacterial transformation. This method is an efficient and convenient technique but it has in some extent low transformation efficiency. Here we report the use of nanoparticles that significantly improve the transformation efficiency up to 10 times higher than the standard heat shock method by the assistance of (˜ 15, 25 nm) SDS capped gold nanoparticles in the transformation process that leads to the formation of temporary nano-channels across the bacterial cell wall to deliver plasmids into cells. Transformation of bacteria with plasmid was examined using Β-galactosidase assay.

scholarly journals HypVW is an HOCl-Sensing Two Component System in Escherichia coli

2021 ◽  
Author(s):  
Sara El Hajj ◽  
Camille Henry ◽  
Alexandra Vergnes ◽  
Laurent Loiseau ◽  
Brasseur Gael ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hypochlorous Acid ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase ◽  
Bacterial Cells ◽  
E Coli ◽  
Two Component ◽  
Redox Switch ◽  
Two Component Systems ◽  
Methionine Residues

Two component systems (TCS) are signalling pathways that allow bacterial cells to sense, respond and adapt to fluctuating environments. Among the classical TCS of Escherichia coli, YedVW has been recently showed to be involved in the regulation of msrPQ, encoding for the periplasmic methionine sulfoxide reductase system. In this study, we demonstrate that hypochlorous acid (HOCl) induces the expression of msrPQ in a YedVW dependant manner, whereas H2O2, NO and paraquat (a superoxide generator) do not. Therefore, YedV appears to be an HOCl-sensing histidine kinase. Based on this finding, we proposed to rename this system HypVW.  Moreover, using a directed mutagenesis approach, we show that Met residues located in the periplasmic loop of HypV (formerly YedV) are important for its activity. Given that HOCl oxidizes preferentially Met residues, we bring evidences that HypV could be activated via the reversible oxidation of its methionine residues, thus conferring to MsrPQ a role in switching HypVW off. Based on these results, we propose that the activation of HypV by HOCl could occur through a Met redox switch. HypVW appears to be the first characterized TCS able to detect HOCl in E. coli. This study represents an important step in understanding the mechanisms of reactive chlorine species resistance in prokaryotes.

Predicting the Behavior of Escherichia coli Introduced as a Postprocessing Contaminant in Shrikhand, a Traditional Sweetened Lactic Fermented Milk Product

2001 ◽  
Vol 64 (4) ◽  
pp. 462-469 ◽  
Author(s):  
A. JAGANNATH ◽  
M. N. RAMESH ◽  
M. C. VARADARAJ
Keyword(s):  
Escherichia Coli ◽  
Response Surface ◽  
Storage Period ◽  
Fermented Milk ◽  
Behavioral Pattern ◽  
Milk Product ◽  
Initial Inoculum ◽  
E Coli ◽  
And Storage ◽  
Fermented Milk Product

The increasing popularity of traditional milk-based foods has placed emphasis on the need for microbial safety in food-chain establishments, as there are ample possibilities for foodborne pathogens to occur as postprocessing contaminants. The behavioral pattern of an enterotoxigenic strain of Escherichia coli D 21 introduced as a postprocessing contaminant in shrikhand, a traditional sweetened lactic fermented milk product, was studied with variables of initial inoculum (4.3, 5.3, and 6.3 log10 CFU/g), storage temperature (4, 10, and 16°C), and storage period (4, 9, and 14 days). During storage of shrikhand prepared individually with Lactobacillus delbruecki ssp. bulgaricus CFR 2028 and Lactococcus lactis ssp. cremoris B-634, there was a steady decrease in the viable count of E. coli that was proportional to the initial inoculum of E. coli introduced into shrikhand. The data were subjected to multivariate analysis, and equations were derived to predict the behavior of E. coli in shrikhand. The predicted values for the probable survival of E. coli showed good agreement with the experimental values with a majority of these predictions being fail-safe. The values of statistical indices showed that the model fits ranged between extremely good and satisfactory. Response surface plots were generated to describe the behavioral pattern of E. coli. The derived models and response surface plots enabled prediction of the survival of E. coli in shrikhand as a function of initial inoculum levels, storage temperatures, and storage periods of shrikhand. These predictions were valid within the limits of the experimental variables used to develop the model.

scholarly journals Transfer of Escherichia Coli to Lemons Slices and Ice during Handling

2017 ◽  
Vol 6 (4) ◽  
pp. 111
Author(s):  
Paul Dawson ◽  
Inyee Han ◽  
Ahmet Buyukyavuz ◽  
Wesam Aljeddawi ◽  
Rose Martinez-Dawson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Room Temperature ◽  
Separate Experiment ◽  
E Coli ◽  
And Storage ◽  
Growth Of Bacteria

The objective of this study was to determine the transfer and survival of bacteria during the handling and storage of lemons and transfer of bacteria during handling of ice. Ice and lemon slices are handled and stored in public eating places and used in beverages. During handling and storage the contamination and growth of bacteria may occur leading to the spread of disease. To fulfill the objective, hands were inoculated with Escherichia coli prior to handling of wet and dry whole lemons and in a separate experiment, ice cubes were handled. E. coli transferred to whole lemons or ice after handling were determined. The CFU per lemon and percentage of E. coli transferred were greater for wet lemons -6123 cfu and 4.62% compared to 469 cfu and .2% for dry lemons. The second experiment found from 2 to 67% of the bacteria on hands were transferred to ice by hands and from 30 to 83% of the bacteria on scoops were transferred to ice. In a third experiment, lemons were inoculated with E. coli, then sliced and stored at 4 or 22C and tested at 0, 4 and 24 hr. Lemons stored at room temperature (22°C) had an increase in E. coli population after 24 hour while those stored under refrigeration had a decrease even though bacteria did survive on lemons in either case. 

Changes in Cell Surface Properties of Shiga Toxigenic Escherichia coli by Quercus infectoria G. Olivier

2009 ◽  
Vol 72 (8) ◽  
pp. 1699-1704 ◽  
Author(s):  
SUPAYANG PIYAWAN VORAVUTHIKUNCHAI ◽  
SAKOL SUWALAK
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Surface Properties ◽  
Cell Surface Hydrophobicity ◽  
Ethanolic Extract ◽  
Bacterial Cells ◽  
Electron Microscopic ◽  
Cell Surface Properties ◽  
E Coli ◽  
Quercus Infectoria

The effects of Quercus infectoria (family Fagaceae) nutgalls on cell surface properties of Shiga toxigenic Escherichia coli (STEC) were investigated with an assay of microbial adhesion to hydrocarbon. The surface of bacterial cells treated with Q. infectoria exhibited a higher level of cell surface hydrophobicity (CSH) toward toluene than did the surface of untreated cells. With 50% ethanolic extract, the CSH of the three strains of STEC O157:H7 treated with 4× MIC of the extract resulted in moderate or strong hydrophobicity, whereas at 2× MIC and MIC, the CSH of only one strain of E. coli O157:H7 was significantly affected. The 95% ethanolic extract had a significant effect on CSH of all three strains at both 4× MIC and 2× MIC but not at the MIC. The effect on bacterial CSH was less pronounced with the other STEC strains. At 4× MIC, the 50% ethanolic extract increased the CSH of all non-O157 STEC strains significantly. At 2× MIC and 4× MIC, the 95% ethanolic extract affected the CSH of E. coli O26:H11 significantly but did not affect E. coli O111:NM or E. coli O22. Electron microscopic examination revealed the loss of pili in the treated cells. The ability of Q. infectoria extract to modify hydrophobic domains enables this extract to partition the lipids of the bacterial cell membrane, rendering the membrane more permeable and allowing leakage of ions and other cell contents, which leads to cell death. Further studies are required to evaluate the effects of Q. infectoria extract in food systems or in vivo and provide support for the use of this extract as a food additive for control of these STEC pathogens.

scholarly journals Utilization of Mn-Doped ZnSe/ZnS Core/Shell Quantum Dots for Rapid Detection of Escherichia coli O157:H7 and Methicillin-Resistant Staphylococcus aureus

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Thi-Diem Bui ◽  
Quang-Liem Nguyen ◽  
Thi-Bich Luong ◽  
Van Thuan Le ◽  
Van-Dat Doan
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Quantum Dots ◽  
Fluorescent Labeling ◽  
Core Shell ◽  
Bacterial Cells ◽  
Bacterial Strains ◽  
Methicillin Resistant ◽  
E Coli ◽  
Mn Doped

In this study, Mn-doped ZnSe/ZnS core/shell quantum dots (CSQDs) were synthesized in aqueous solution using polyethylene glycol as a surface stabilizer and successfully applied in the detection of Escherichia coli O157:H7 and methicillin-resistant Staphylococcus aureus (MRSA) for the first time. The CSQDs were conjugated with anti-E. coli antibody and anti-MRSA antibody via protein A supported by 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride for fluorescent labeling of the intact bacterial cells. The detection was performed for the bacterial strains cultivated in Luria-Bertani liquid medium. The obtained results indicate that E. coli O157:H7 and MRSA can be detected within 30 min at a high sensitivity of 101 CFU/mL. This labeling method based on the highly fluorescent CSQDs may have great potential for use in the food industry to check and prevent outbreaks of foodborne illness.

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