Journal: Biosensors

Article Title: Simultaneous Detection of Salmonella typhimurium and Escherichia coli O157:H7 in Drinking Water and Milk with Mach–Zehnder Interferometers Monolithically Integrated on Silicon Chips

doi: 10.3390/bios12070507

Figure Lengend Snippet: ( a ) Percent signal values obtained for a calibrator containing 25 (yellow columns) or 250 ng/mL of E. coli LPS (orange columns) with respect to zero calibrator (red columns) without pre-incubation and with 5-, 10-, 15-, 30- and 60-min pre-incubation of the calibrators with anti- E. coli antibody solution. ( b ) Percent signal values obtained for a calibrator containing 25 (cyan columns) or 250 ng/mL of S. typhimurium LPS (light blue columns) with respect to zero calibrator (blue columns) without pre-incubation and with 5-, 10-, 15-, 30- and 60-min pre-incubation of the calibrators with anti- S. typhimurium antibody solution. Each point is the mean value of three measurements. Error bars correspond to ±SD.

Article Snippet: Salmonella enterica serovar typhimurium ( S. typhimurium , ATCC 14028), Escherichia coli O157:H7 ( E. coli O157:H7 , NCTC 12900), Escherichia coli ( TOP10 Competent cells clone 2 , ATCC PTA-5689), and Salmonella enterica serovar Thomson were kindly provided from Delta Foods S.A. (Athens, Greece).

Techniques: Incubation

Journal: Biosensors

Article Title: Simultaneous Detection of Salmonella typhimurium and Escherichia coli O157:H7 in Drinking Water and Milk with Mach–Zehnder Interferometers Monolithically Integrated on Silicon Chips

doi: 10.3390/bios12070507

Figure Lengend Snippet: ( a ) Real-time response obtained for S. typhimurium zero calibrator prepared in assay buffer (arrow 1 to 4) or in bottled water (arrow 7 to end). The arrows indicate the sequence of solutions passing over the chip: assay buffer (start to 1); zero calibrator in assay buffer (arrow 1 to 2); biotinylated anti-species specific antibody (arrow 2 to 3); streptavidin (arrow 3 to 4); 50 mM HCl (arrow 4 to 5); 50 mM NaOH (arrow 5 to 6); assay buffer (arrow 6 to 7); zero calibrator in bottled water (arrow 7 to 8); biotinylated anti-species specific antibody (arrow 8 to 9); streptavidin (arrow 9 to end). ( b ) Real-time response obtained for E. coli zero calibrator prepared in milk. The arrows indicate the sequence of solutions passing over the chip: assay buffer (arrow 1 to 2), zero calibrator prepared in milk (arrow 2 to 3), washing buffer (arrow 3 to 4), assay buffer (arrow 4 to 5), biotinylated anti-species-specific antibody (arrow 5 to 6) and streptavidin (arrow 6 to end). Dashed line corresponds to non-specific binding signal.

Article Snippet: Salmonella enterica serovar typhimurium ( S. typhimurium , ATCC 14028), Escherichia coli O157:H7 ( E. coli O157:H7 , NCTC 12900), Escherichia coli ( TOP10 Competent cells clone 2 , ATCC PTA-5689), and Salmonella enterica serovar Thomson were kindly provided from Delta Foods S.A. (Athens, Greece).

Techniques: Sequencing, Binding Assay

Journal: Biosensors

Article Title: Simultaneous Detection of Salmonella typhimurium and Escherichia coli O157:H7 in Drinking Water and Milk with Mach–Zehnder Interferometers Monolithically Integrated on Silicon Chips

doi: 10.3390/bios12070507

Figure Lengend Snippet: Calibration curves obtained for ( a ) E. coli and ( b ) S. typhimurium with calibrators prepared using live untreated (grey squares), heat-treated (blue triangles), ultra-sonicated (red circles) or heat-treated and ultra-sonicated bacteria (green triangles). Each point is the mean value of three measurements. Error bars correspond to ±SD.

Article Snippet: Salmonella enterica serovar typhimurium ( S. typhimurium , ATCC 14028), Escherichia coli O157:H7 ( E. coli O157:H7 , NCTC 12900), Escherichia coli ( TOP10 Competent cells clone 2 , ATCC PTA-5689), and Salmonella enterica serovar Thomson were kindly provided from Delta Foods S.A. (Athens, Greece).

Techniques: Sonication, Bacteria

Journal: Biosensors

Article Title: Simultaneous Detection of Salmonella typhimurium and Escherichia coli O157:H7 in Drinking Water and Milk with Mach–Zehnder Interferometers Monolithically Integrated on Silicon Chips

doi: 10.3390/bios12070507

Figure Lengend Snippet: Calibration curves of ( a ) S. typhimurium and ( b ) E. coli . (S x /S 0 ) × 100 represents the percent ratio of each calibrator signal (S x ) to the zero calibrator signal (S 0 ). Each point is the mean value of three chips ±SD.

Article Snippet: Salmonella enterica serovar typhimurium ( S. typhimurium , ATCC 14028), Escherichia coli O157:H7 ( E. coli O157:H7 , NCTC 12900), Escherichia coli ( TOP10 Competent cells clone 2 , ATCC PTA-5689), and Salmonella enterica serovar Thomson were kindly provided from Delta Foods S.A. (Athens, Greece).

Techniques:

Journal: Biosensors

Article Title: Simultaneous Detection of Salmonella typhimurium and Escherichia coli O157:H7 in Drinking Water and Milk with Mach–Zehnder Interferometers Monolithically Integrated on Silicon Chips

doi: 10.3390/bios12070507

Figure Lengend Snippet: ( a ) Calibration curves for S. typhimurium (black squares), E. coli O157:H7 (red circles), E. coli top10 (blue triangles) and S . Thomson (green hexagones) obtained from MZIs spotted with S. typhimurium LPS. ( b ) Calibration curves for E. coli O157:H7 (black squares), E. coli top10 (red circles), S. typhimurium (blue triangles) and S. Thomson (green hexagones) obtained from MZIs spotted with E. coli LPS. The dashed vertical lines indicate the bacteria concentration that corresponds to a 50% signal drop with respect to zero calibrator (horizontal black line). Each point is the mean value of seven waveguides per chip ± SD.

Article Snippet: Salmonella enterica serovar typhimurium ( S. typhimurium , ATCC 14028), Escherichia coli O157:H7 ( E. coli O157:H7 , NCTC 12900), Escherichia coli ( TOP10 Competent cells clone 2 , ATCC PTA-5689), and Salmonella enterica serovar Thomson were kindly provided from Delta Foods S.A. (Athens, Greece).

Techniques: Bacteria, Concentration Assay

Journal: Biosensors

Article Title: Simultaneous Detection of Salmonella typhimurium and Escherichia coli O157:H7 in Drinking Water and Milk with Mach–Zehnder Interferometers Monolithically Integrated on Silicon Chips

doi: 10.3390/bios12070507

Figure Lengend Snippet: Real-time immunosensor responses when running a zero calibrator over the chip prepared in 10% milk in peptone water (purple line) and a milk sample initially containing 1 cfu of E. coli /25 g milk (pink line) after 7.5 h pre-enrichment. The arrows indicate the sequence of solutions passing over the chip: assay buffer (arrow 1 to 2), zero calibrator/sample in 10% milk in peptone water (arrow 2 to 3), washing (arrow 3 to 4), assay buffer (arrow 4 to 5), biotinylated anti-species-specific antibody (arrow 5 to 6) and streptavidin (arrow 6 to end). Dashed line corresponds to non-specific binding signal.

Article Snippet: Salmonella enterica serovar typhimurium ( S. typhimurium , ATCC 14028), Escherichia coli O157:H7 ( E. coli O157:H7 , NCTC 12900), Escherichia coli ( TOP10 Competent cells clone 2 , ATCC PTA-5689), and Salmonella enterica serovar Thomson were kindly provided from Delta Foods S.A. (Athens, Greece).

Techniques: Sequencing, Binding Assay

Journal: Biosensors

Article Title: Simultaneous Detection of Salmonella typhimurium and Escherichia coli O157:H7 in Drinking Water and Milk with Mach–Zehnder Interferometers Monolithically Integrated on Silicon Chips

doi: 10.3390/bios12070507

Figure Lengend Snippet: Comparison of the proposed MZI immunosensor with other optical label-free immunosensors.

Article Snippet: Salmonella enterica serovar typhimurium ( S. typhimurium , ATCC 14028), Escherichia coli O157:H7 ( E. coli O157:H7 , NCTC 12900), Escherichia coli ( TOP10 Competent cells clone 2 , ATCC PTA-5689), and Salmonella enterica serovar Thomson were kindly provided from Delta Foods S.A. (Athens, Greece).

Techniques: Comparison, Bacteria, Immunomagnetic Separation, Imaging

Journal: Nature Communications

Article Title: Overcoming the design, build, test bottleneck for synthesis of nonrepetitive protein-RNA cassettes

doi: 10.1038/s41467-021-21578-6

Figure Lengend Snippet: a (Top) Wild-type binding sites for MS2, PP7, and Qβ phage coat proteins and illustrations of the 20k mutated variants created based on their sequences. (Bottom) Composition of the OL library. Histogram of the number of PP7-based variants (blue), Qβ-based variants (orange), and MS2-based variants (green) with different edit distances from the MS2-WT binding site. b Each putative binding site variant was encoded on a 210 bp oligo containing the following components: restriction site, barcode, constitutive promoter (cPr), ribosome binding site (RBS), mCherry start codon, one or two bases (denoted by δ), the sequence of the variant tested, and the second restriction site. Each configuration was encoded with five different barcodes, resulting in a total of 100k different OL variants. The OL was then cloned into a vector and transformed into an E. coli strain expressing one of three RBP–GFP fusions under an inducible promoter (iPr). The transformation was repeated for all three fusion proteins. c The schema illustrates the behavior of a high-affinity strain: when no inducer is added, mCherry is expressed at a certain basal level that depends on the mRNA structure and sequence. When inducer (C4-HSL) is added, the RBP binds the mRNA and blocks the ribosome from mCherry translation, resulting in a down-regulatory response as a function of inducer concentration. d The experimental flow for iSort-seq. Each library is grown at six different inducer concentrations, and sorted into eight bins with varying mCherry levels and constant RBP–GFP levels. This yields a 6 × 8 matrix of mCherry levels for each variant at each induction level. (Bottom) An illustration of the experimental output of a high-affinity strain (V1) and a no-affinity strain (V2). See Fig. for flow cytometry gating strategy details.

Article Snippet: Then, we performed transformation to Top10 E. coli cells (Lucigen) and screened for positive clones.

Techniques: Binding Assay, Variant Assay, Sequencing, Clone Assay, Plasmid Preparation, Transformation Assay, Expressing, Concentration Assay, Flow Cytometry

Journal: Nature Communications

Article Title: Overcoming the design, build, test bottleneck for synthesis of nonrepetitive protein-RNA cassettes

doi: 10.1038/s41467-021-21578-6

Figure Lengend Snippet: a R score comparison to ΔG results of a previous study that reported MCP binding to more than 129k sequences . Each plot (from left to right) represents Pearson correlation coefficient using: the experimental measurements for variants that were both in our OL and in the in vitro study, the R score values predicted by our ML model for all single-mutation variants, for all double-mutation variants, and for the entire set of 129,248 mutated variants. b Example experiment design for bacterial and mammalian cells. High R score binding sites were incorporated into a ten-site cassette downstream to either a T7 promoter in E. coli or a mammalian CMV promoter. When the matching QCP-mCherry for bacteria (top) or QCP-3xBFP for mammalian cells (bottom) are added, it binds the binding site cassette and creates a fluorescent spot. c Fluorescent image of typical bacterial cell depicting bright puncta in cell pole. (Left) Raw image. (Right) Processed image depicting variations in brightness density. d Violin plots of mean spot intensity measured in bacteria expressing the different binding sites cassettes. Values are based on the following number of captured cells taken on multiple days (from left to right): 99 (PP7-4x), 149 (Qβ-5x), 187 (Qβ-10x), 182 (PP7-24x). e The results for three CP-specific cassettes transfected with the matching CP-3xFP plasmid into U2OS cells and imaged by fluorescence microscopy for detection of fluorescent foci . For each experiment, both the relevant fluorescent channel and the merged images with the differential interference contrast (DIC) channel are presented. Each experiment was successfully conducted in duplicates and on two different days. f (Left) Experimental design for the orthogonality experiment: two separate cassettes with 10 predicted mutated sites for either MCP only or QCP only, respectively, were designed and transfected together with both MCP-3xmCherry and QCP-3xBFP, into U2OS cells. (Right) Results for the orthogonality experiment: a cell presenting non-overlapping fluorescent foci from both fluorescent channels, indicating binding of MCP and QCP to different targets. This experiment was successfully conducted in duplicate and on two different days. Fluorescent wavelengths used in these experiments are: 400 nm for BFP, 490 nm for GFP, and 585 nm for mCherry. Source data are provided as a Source data file.

Article Snippet: Then, we performed transformation to Top10 E. coli cells (Lucigen) and screened for positive clones.

Techniques: Comparison, Binding Assay, In Vitro, Mutagenesis, Bacteria, Expressing, Transfection, Plasmid Preparation, Fluorescence, Microscopy