Journal: Biosensors

Article Title: Microbead-Encapsulated Luminescent Bioreporter Screening of P. aeruginosa via Its Secreted Quorum-Sensing Molecules

doi: 10.3390/bios14080383

Figure Lengend Snippet: Schematic of the cellular events that result in the expression of a reporter protein. The cell can easily take up a bioavailable analyte, such as AHL, through its membrane. AHL (3-oxo-C 12 -HSL and C 4 -HSL) binds with a regulatory protein called LasR or RhlR, forming a protein–AHL complex. This complex binds to its corresponding promoter, Plasl or Prhl , which triggers activation of the transcription and translation of a reporter gene called luxCDABE. This results in the expression of the reporter protein, luciferase, inducing the bioluminescent reaction characterized by the emission of blue-green light (~490 nm). Created with BioRender.com, accessed on 13 June 2024.

Article Snippet: Acyl-homoserine lactone (acyl-HSL) 3-oxo-C 12 -HSL, C 4 -HSL, C 6 -HSL, 3-oxo-C 8 -HSL, 3-oxo-C 6 -HSL, and (as in ) the stock (5 mM) solutions were stored at −20 °C until required, and, as well as QS inhibitor—furanone C-30, were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Expressing, Membrane, Activation Assay, Luciferase

Journal: Biosensors

Article Title: Microbead-Encapsulated Luminescent Bioreporter Screening of P. aeruginosa via Its Secreted Quorum-Sensing Molecules

doi: 10.3390/bios14080383

Figure Lengend Snippet: The bioluminescent response of the immobilized RhlR in different C 4 -HSL concentrations. The insert in ( A ) illustrates the induction factor calculated using the maximum relative luminescence unit (RLU) derived from the spectral data. The induction factor is the ratio of the test’s maximum RLU conducted in the presence of an inducer to the maximum RLU obtained in the absence of added inducers. ( B ) Confocal images of the live/dead stained bacteria within alginate-PLL beads. ( C ) Both the luminescence and cell density (absorbance at 600 nm) of the LasR bioreporter. ( D ) The luminescence and OD 600 readings of the RhlR bioreporter. The OD 600 -normalized luminescence reading (RLU/OD 600 ) can be found in .

Article Snippet: Acyl-homoserine lactone (acyl-HSL) 3-oxo-C 12 -HSL, C 4 -HSL, C 6 -HSL, 3-oxo-C 8 -HSL, 3-oxo-C 6 -HSL, and (as in ) the stock (5 mM) solutions were stored at −20 °C until required, and, as well as QS inhibitor—furanone C-30, were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Derivative Assay, Staining, Bacteria

Journal: Biosensors

Article Title: Microbead-Encapsulated Luminescent Bioreporter Screening of P. aeruginosa via Its Secreted Quorum-Sensing Molecules

doi: 10.3390/bios14080383

Figure Lengend Snippet: The bioluminescent responses of the PLL- and PDL-coated alginate were similar. The PDL and PLL coatings were compared regarding the bacteria’s response to the added synthetic (C 4 -HSL) and secreted (by the PAO1 wild strain of P. aeruginosa ) autoinducers.

Article Snippet: Acyl-homoserine lactone (acyl-HSL) 3-oxo-C 12 -HSL, C 4 -HSL, C 6 -HSL, 3-oxo-C 8 -HSL, 3-oxo-C 6 -HSL, and (as in ) the stock (5 mM) solutions were stored at −20 °C until required, and, as well as QS inhibitor—furanone C-30, were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques:

Journal: Biosensors

Article Title: Microbead-Encapsulated Luminescent Bioreporter Screening of P. aeruginosa via Its Secreted Quorum-Sensing Molecules

doi: 10.3390/bios14080383

Figure Lengend Snippet: Calibration curve of the RlhR bioreporter beads at various concentrations of C 4 -HSL. The left panel shows the bioluminescence (expressed as an induction factor) produced by the encapsulated reporter in the presence of increasing concentrations of exogenously added C 4 -HSL. The right panel shows the dose–response relationships inferred from the data in the left panel. Linear regression lines (bold) were drawn on logarithmic and linear (upper insert) scales, and the 95% confidence interval limits were the black dotted lines parallel to the regression lines. The equation and the R 2 value for each regression line are shown. Data are the mean ± SD of three independent experiments.

Article Snippet: Acyl-homoserine lactone (acyl-HSL) 3-oxo-C 12 -HSL, C 4 -HSL, C 6 -HSL, 3-oxo-C 8 -HSL, 3-oxo-C 6 -HSL, and (as in ) the stock (5 mM) solutions were stored at −20 °C until required, and, as well as QS inhibitor—furanone C-30, were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Produced

Journal: Biosensors

Article Title: Microbead-Encapsulated Luminescent Bioreporter Screening of P. aeruginosa via Its Secreted Quorum-Sensing Molecules

doi: 10.3390/bios14080383

Figure Lengend Snippet: The bioreporter specificity test. The RhlR ( A ) and LasR ( B ) response toward 5 µM each of synthetic QS molecules, respectively. The second experiment was conducted with either 20 µL of cell-free supernatants of each Gram-negative bacterium such as Escherichia coli ( E. coli ), Pseudomonas aeruginosa (PAO1), and Acinetobacter baumannii ( A. baumannii ) (72 h biofilm set up), or 5 µM of C 4 -HSL for the RhlR strain ( C ) or 5 µM of 3-oxo-C 12 -HSL for the LasR strain ( D ).

Article Snippet: Acyl-homoserine lactone (acyl-HSL) 3-oxo-C 12 -HSL, C 4 -HSL, C 6 -HSL, 3-oxo-C 8 -HSL, 3-oxo-C 6 -HSL, and (as in ) the stock (5 mM) solutions were stored at −20 °C until required, and, as well as QS inhibitor—furanone C-30, were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques:

Journal: Biosensors

Article Title: Microbead-Encapsulated Luminescent Bioreporter Screening of P. aeruginosa via Its Secreted Quorum-Sensing Molecules

doi: 10.3390/bios14080383

Figure Lengend Snippet: The QS inhibitory effect of furanone C-30 in the presence of synthetic AHLs. ( A ) The inhibition of RhlR signaling in the presence of 5 µM of C 4 -HSL and ( B ) the inhibition of the LasR bioluminescence system in the presence of 5 µM of 3-OC 12 -HSL. Luminescent readings were taken for 10 h, and the maximum relative light unit (RLU) was plotted. The results are the mean ± SD of biological triplicates.

Article Snippet: Acyl-homoserine lactone (acyl-HSL) 3-oxo-C 12 -HSL, C 4 -HSL, C 6 -HSL, 3-oxo-C 8 -HSL, 3-oxo-C 6 -HSL, and (as in ) the stock (5 mM) solutions were stored at −20 °C until required, and, as well as QS inhibitor—furanone C-30, were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Inhibition

Journal: mBio

Article Title: Temperature, by Controlling Growth Rate, Regulates CRISPR-Cas Activity in Pseudomonas aeruginosa

doi: 10.1128/mBio.02184-18

Figure Lengend Snippet: QS and low temperature act synergistically to enhance CRISPR-Cas-mediated adaptation. PCR amplification of the CRISPR2 array and visualization by gel electrophoresis, as in Fig. 1 . The PA14 Δ lasI Δ rhlI mutant does not produce 3OC 12 -HSL or C 4 -HSL. 3OC 12 -HSL and C 4 -HSL (designated AI) were supplied at saturating concentrations (2 μM and 10 μM, respectively), as denoted. The data are representative of >3 independent experiments.

Article Snippet: For AI supplementation assays, 3OC 12 -HSL and C 4 -HSL (Sigma) or the solvent dimethyl sulfoxide (DMSO) was used.

Techniques: CRISPR, Amplification, Nucleic Acid Electrophoresis, Mutagenesis