The fluorescent d -amino acid (FDAA) NADA (4-chloro-7-nitrobenzofurazan 3-amino-d -alanine) or TADA (tetramethylrhodamine 3-amino-d -alanine) (18 (link), 51 (link)) was added to early exponential cells (OD600 of 0.05) to a final concentration of 100 μM. Cultures were grown to an OD600 of 0.20 and placed on ice for 2 min to halt labeling. One milliliter of culture was centrifuged at 14,000 × g for 5 min at 4°C. Cell pellets were washed once with cold medium, resuspended, and seeded into microfluidic chambers at 10°C. Flow was then initiated with prewarmed medium without peptide (control) or with the indicated concentration of peptide (treatment). Where indicated, expression of a FliM-tdTomato fusion (10 (link)) was induced with 0.1% l -Ara (Sigma-Aldrich), and cells were stained with 4′,6′-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 5 min prior to the addition of 6-carboxyfluorescein (FAM)-labeled B22.
L ara
Manufactured by Merck Group
Sourced in United States
L-Ara is a laboratory equipment designed for the analysis and quantification of L-arabinose, a naturally occurring sugar. The core function of L-Ara is to provide accurate and reliable measurements of L-arabinose levels in various samples, enabling researchers and scientists to study its properties and applications.
Automatically generated - may contain errors
Lab products found in correlation
D gal, by Merck Group (5 mentions)
D glc, by Merck Group (4 mentions)
D xyl, by Merck Group (3 mentions)
D glca, by Merck Group (3 mentions)
L fuc, by Merck Group (3 mentions)
L rha, by Merck Group (3 mentions)
D gala, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Dionex ics 5000, by Thermo Fisher Scientific (1 mentions)
Ix83 inverted microscope, by Olympus (1 mentions)
Gibson assembly kit, by New England Biolabs (1 mentions)
Cellsense dimensions v 1.14, by Olympus (1 mentions)
Orca r2, by Hamamatsu Photonics (1 mentions)
Carbopac pa20 column, by Thermo Fisher Scientific (1 mentions)
Carbopac pa1 column, by Thermo Fisher Scientific (1 mentions)
Hpaec pad, by Thermo Fisher Scientific (1 mentions)
Ultraspec 8000, by GE Healthcare (1 mentions)
D fru, by Merck Group (1 mentions)
Pa20 column, by Thermo Fisher Scientific (1 mentions)
8 protocols using l ara
1
Fluorescent D-Amino Acid Labeling for Bacterial Imaging
2
Quantifying Non-Cellulosic Polysaccharides
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The composition of non-cellulosic polysaccharides was determined according to Foster et al. (2010) with modifications. Due to shortage of plant material, analysis was performed only for samples harvested at the heading stage and maturity. De-starched AIR material (5 mg) was hydrolysed in 2 M TFA for 90 min at 121 °C. TFA was removed by drying under vaccum. TFA breaks down the cell wall network to release primarily non-cellulosic polysaccharides, and some fractions of cellulose that contain kinks and chain dislocations (amorphous form). The composition of monosaccharides in the filtrate was determined and quantified by high-performance anion-exchange chromatography coupled with integrated pulsed amperometric detection (HPAEC-iPAD) using Thermo Scientific Dionex ICS5000 as described previously by Głazowska et al. (2018) (link). The system was calibrated with standards (l -Ara, d -Xyl, d -Gal, d -Glc, d -GlcA, and d -GalA) (Sigma). All calculations were done in Chromeleon CDS software.
3
Microscopic Visualization of Bacterial Motility
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Single-cell static images and movies were captured on an Olympus IX83 inverted microscope using a 100×, 1.3-numerical-aperture phase-contrast objective. Fluorescence images were obtained with a Hamamatsu ORCA-R2 digital charge-coupled-device camera, and the light source was the Xcite 120 light-emitting diode (Lumen Dynamics, Mississauga, Ontario, Canada). Emission filters were purchased from Chroma Technology (Bellows Falls, VT). Specific emission filters were DAPI-5060C-OMF (excitation [EX] filter, 377/50 nm; emission [EM] filter, 447/60 nm; dichroic mirror [DM], 409 nm), GFP-3035D-OMF (EX filter, 473/31 nm; EM filter, 520/35 nm; DM, 495 nm), mCherry-B-OFF (EX filter, 562/40 nm; EM filter, 641/75 nm; DM, 593 nm). Images were processed with the Olympus software package cellSense Dimensions (v 1.14). The V. vulnificus fliM-tdTomato fusion was cloned into pSU38 (27 (link)) using the Gibson assembly kit (New England Biolabs, Ipswich, MA), and expression was induced with 0.1% l -Ara (Sigma-Aldrich). Where indicated, cells were stained with DAPI for 5 min prior to the addition of 6-carboxyfluorescein (FAM)−SMAP-29D.
4
Monosaccharide Composition Analysis of Plant Cell Walls
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To determine monosaccharide composition, 3 replicates of cell walls from 10 plants were used. 1 mg of dry de-starched cell wall was hydrolyzed with 2 N trifluoroacetic acid at 120 °C for 2 h. The hydrolysates were dried at 50 °C, re-dissolved in water, and analyzed by high-performance anion-exchange chromatography with pulsed-amperometric detection using a CarboPac PA-20 column (3 mm × 150 mm; Dionex, Sunnyvale, CA, USA) as described earlier [99 (link)]. Monosaccharides were separated using a gradient of 100 mM NaOH in water at 0.5 mL min−1 under the following conditions: 0–0.05 min—12 mM NaOH; 0.05–26 min—0.65 mM NaOH; 26–46 min—300 mM NaOH; 46–55 min—12 mM NaOH. Monosaccharide standards included L-Fuc, L-Rha, L-Ara, D-Gal, D-Glc, D-Xyl, D Man, D-GalA, and D-GlcA (all from Sigma–Aldrich, St. Louis, MO, USA). To determine response factors, standard curves were created using mixtures of all standard monosaccharides at different concentrations.
5
Genetic Circuit Construction in E. coli
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DH10B and K-12 MG1655ΔlacIΔaraCBAD strains of Escherichia coli were utilized for gene circuit construction process and experimentation respectively. Cells were grown in 15 mL tubes at 37C on a shaker set to 220 RPM rotational speed in Luria-Bertani (LB) medium containing either 25 μg ml−1 chloramphenicol, 100 μg ml−1 ampicillin, or 50 μg ml−1 kanamycin. L-ara (Sigma-Aldrich) and aTc (Abcam) were dissolved into stock concentrations of 25% (volume-wise) and 1000 ng/ul respectively, then diluted with ddH2O into 1000× working solutions before being utilized for experimentation. Diluted media utilized to create the moderate growth condition during hysteresis experiments was created by mixing LB with M9 minimal media.
6
Sequential Extraction of CW Polysaccharides
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The sequential extraction of CW polysaccharides was performed in four steps and detailed in [16 (link)]. In summary, 100 mg of a deproteinized CW fraction were used. Four successive extractions were carried out to obtain extracts enriched in pectins (E1 and E2, respectively using CDTA 50 mM and 50 mM Na2CO3) and hemicelluloses (E3 and E4, respectively using 20 mM NaBH4 and 4 M NaOH). Each extract was hydrolyzed in 2 N TFA for 1 h at 120 °C. After 10× dilution in UHQ water, monosaccharides were analysed by High-Performance Anion-Exchange Chromatography coupled to Pulsed Amperometric Detection (HPAEC-PAD; Dionex, Sunnyvale, California, USA) using a CarboPac PA1 column (Dionex). L-Fuc, L-Rha, L-Ara, D-Gal and GalA (Sigma-Aldrich); D-Glc (Merck, Darmstadt, Germany); D-Xyl (Roche, Mannheim, Germany) were used as standard monosaccharides for identification and quantification. Data are described in [43 (link)]. CW polysaccharides reconstruction was performed using formula previously described [16 (link),43 (link)] and adapted from [47 (link)].
7
Carbon Utilization of E. coli Strains
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The E. coli strains, plasmids, and primers used in this study are listed in Tables S2 to S4. Cultures were grown in Luria-Bertani (LB) medium and minimal medium (M9) at 37°C with kanamycin (Kan) at 50 μg mL−1 or ampicillin (Amp) at 100 μg mL−1. E. coli DH5α was used as a bacterial host for cloning, and E. coli BL21(DE3) was used for protein expression. For carbon utilization study, E. coli strains were grown aerobically in minimal medium (M9) supplemented with 0.5% (wt/vol) of each carbon source (e.g., d- Glc, d- Fru, d- Gal, l -Ara, and d- Tag; Sigma, St. Louis, MO, USA) at 37°C, harvested by centrifugation at 1,500 × g for 20 min, washed twice with M9 medium without a carbon source, and resuspended in appropriate fresh medium. Inoculated cultures (OD600 of 0.05) were grown aerobically at 37°C, and bacterial growth was monitored by measuring absorbance at 600 nm (OD600) with an Ultraspec 8000 spectrophotometer (GE Healthcare, Piscataway, NJ, USA). Unless otherwise stated, bacterial culture experiments were performed in triplicate.
8
Monosaccharide Profiling by HPAEC-PAD
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Cell wall monosaccharide was determined by HPAEC-PAD of hydrolysed material. A PA20 column (Dionex, USA) was used at a flow rate of 0.5 ml/min. The column was washed with 200 mM NaOH for 10 min before the injection of each sample and then equilibrated with 10 mM NaOH for 10 min.
The elution programme consisted of an isocratic elution with 10 mM NaOH from 0 to 37 min, followed by a linear gradient up to 800 mM NaOH from 37 to 43 min, and finally down to 10 mM NaOH from 43 to 45 min. Monosaccharide standards included L-Fuc, L-Rha, L-Ara, D-Gal, D-Glc, D-Xyl, D-Man, D-GalUA and D-GlcA (Sigma, UK); the standard mixture concentration ranges from 0.001 μg/10 μl to 0.1 μg/10 μl. A standard mixture run was performed before analysis of a batch of samples for verification of the response factors.
The elution programme consisted of an isocratic elution with 10 mM NaOH from 0 to 37 min, followed by a linear gradient up to 800 mM NaOH from 37 to 43 min, and finally down to 10 mM NaOH from 43 to 45 min. Monosaccharide standards included L-Fuc, L-Rha, L-Ara, D-Gal, D-Glc, D-Xyl, D-Man, D-GalUA and D-GlcA (Sigma, UK); the standard mixture concentration ranges from 0.001 μg/10 μl to 0.1 μg/10 μl. A standard mixture run was performed before analysis of a batch of samples for verification of the response factors.
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