The β-galactosidase assay in perspective: Critical thoughts for biosensor development

Hosseini, Anahita; Mas, Jordi

doi:10.1016/j.ab.2021.114446

Cited by 5 publications

(3 citation statements)

References 49 publications

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“…β-galactosidase is a hydrolase located on the bacterial cytoplasmic membrane. Detection of β-galactosidase in the supernatant can evaluate the effects of a drug on the cell membrane permeability [ 36 ]. In the present study, LL-1 was able to increase the permeability of E. coli cell membrane, resulting in leakage of intracellular β-galactosidase, nucleic acids and proteins.…”

Section: Discussionmentioning

confidence: 99%

The antimicrobial effect of a novel peptide LL-1 on Escherichia coli by increasing membrane permeability

et al. 2022

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Background The widespread use of antibiotics has led to the emergence of many drug-resistant strains; thus, the development of new antibacterial drugs is essential with antimicrobial peptides becoming the focus of research. This study assessed the antibacterial effect of a novel antimicrobial peptide, named LL-1 on Escherichia coli (E.coli) by determining the minimum inhibitory concentration (MIC) and the antibacterial curve. The interaction between LL-1 and E. coli DNA was then detected by nucleic acid gel electrophoresis. The effect of LL-1 on the E. coli cell membrane was assessed by detecting the leakage of β-galactosidase, nucleic acid and protein. The influence of LL-1 on the intracellular ATP of E. coli was analysed by determining the concentration of intracellular ATP. Finally, the bacteria and colonies of E. coli treated with LL-1 were observed using scanning and transmission electron microscopy. Results The results suggested that the MIC value was 3.125 µg/ml, and the antibacterial effect was dose-dependent. LL-1 dose-dependently combined with E. coli DNA. LL-1 resulted in the leakage of intracellular β-galactosidase, nucleic acid and protein, and decreased intracellular ATP concentrations of E. coli. Two MIC of LL-1 caused E. coli to shrink, resulting in a rough surface, plasmolysis, and bacterial adhesion. Conclusion This study indicated that LL-1 had a good bactericidal effect on E. coli by mainly increasing the permeability of the cell membrane, leading to leakage of the intracellular content. This will lay the foundation for an in-depth study on the antibacterial mechanism of LL-1 against E. coli and its clinical application.

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Section: Discussionmentioning

confidence: 99%

The antimicrobial effect of a novel peptide LL-1 on Escherichia coli by increasing membrane permeability

et al. 2022

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“…Laboratory standards of E.coli for the calibration of the assay were prepared inoculating 0.2 mL of an overnight culture in 5 mL of fresh LB medium and incubating under aerobic conditions at 37 o C for approximately 1 hour until reaching the middle of the stationary phase (OD 600 of 0.4-0.5). At that point the culture was supplemented with 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) and incubated for a further 3 hours to fully induce the expression of ß-galactosidase and maximize the enzyme content [22]. After induction the culture was centrifuged at 4000 g for 10 minutes and then resuspended in PBS buffer.…”

Section: Preparation Of E Coli Standardsmentioning

confidence: 99%

“…The method used for the ß-galactosidase assay is an adaptation of the 96 well plate method described by Schaeffer et al [15] as modi ed by Hosseini and Mas [22]. To carry out the assay, 50 µL of the appropriate dilution of a fully induced E. coli culture were added to a microplate well containing 120 µL Zbuffer (60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl, 1 mM MgSO 4 , 50 mM ß-mercaptoethanol, pH = 7), 20 µL of a 10 9 pfu•mL − 1 phage solution and 10 µL PBS.…”

Section: Microplate-based ß-Galactosidase Assaymentioning

confidence: 99%

Specific detection of Escherichia coli using a phage-assisted ß-galactosidase assay

Hosseini

Mas

2023

Preprint

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Fast and reliable detection of microbial contaminants in food, water and environmental samples is critical for an efficient public health management. Most available methods provide good results although many of them have a number of drawbacks ranging from low sensitivity to the need of sophisticated equipment, the use of expensive reagents or the participation of highly skilled personnel. This work describes an easy to implement method for the detection of E. coli in liquid samples using a robust non-specific ß-galactosidase assay made highly selective through the use of a specific T4 lytic phage as a permeabilization reagent. The assay is performed in 96 well plates using MUG (4-methylumberlliferyl-ß-D-galactopyranoside) as the enzyme substrate and has a total length of 90 minutes. The method is able to detect 75 cells of E. coli. Under the conditions of the assay this corresponds to a concentration of 1.49·103 cells·mL− 1 of sample. For the analysis of field samples, we produced an extended version of the assay that incorporates preconcentration and preincubation steps with a total running length of 7.5 hours. When tested with field samples and compared with Colilert-18 the method performed well, with a limit of detection of 96 cells·100 mL− 1.

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GH2 family β-galactosidases evolution using degenerate oligonucleotide gene shuffling

2023

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The β-galactosidase assay in perspective: Critical thoughts for biosensor development

Cited by 5 publications

References 49 publications

The antimicrobial effect of a novel peptide LL-1 on Escherichia coli by increasing membrane permeability

The antimicrobial effect of a novel peptide LL-1 on Escherichia coli by increasing membrane permeability

Specific detection of Escherichia coli using a phage-assisted ß-galactosidase assay

GH2 family β-galactosidases evolution using degenerate oligonucleotide gene shuffling

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