Lateral flow detection of iSDA amplicons was by a twin probe method co-developed with our colleagues at ELITechGroup.39 (link) In short, a 3′-biotinylated detection probe hybridizes to the amplicon and binds a streptavidin-coated Au nanoparticle label. A chimeric pyranosyl DNA (pDNA)–DNA capture probe hybridizes by DNA independently to the amplicon and hybridizes by pDNA to an immobilized pDNA complement. A dipstick-style LF assay format was used for demonstrating the dry preservation of the iSDA reagents. Cardboard-backed nitrocellulose FF80HP (GE Healthcare, Waukesha, WI) striped with (1) a pDNA complement linked to T20 (twenty repeats of thymidine) and (2) a T20-biotin control line (provided by ELITechGroup, Bothell, WA), and attached to a cellulose wicking pad, was cut into ~5 mm-wide strips. For detection, 28 μl of the iSDA reaction mix containing amplicons was mixed with a solution of NaCl and Triton-X100 to give final concentrations of 0.6 M and 0.8%, respectively. The mixture was added into a 96-well deep well plate (VWR, Radnor, PA) and a detection strip placed into each well for 20 minutes. The LF strips were then imaged using the procedure described below.
96 well deep well plate
Manufactured by Avantor
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96-well deep well plates are laboratory equipment used for sample preparation, storage, and processing. They provide a 96-well format with increased well depth compared to standard microplates, allowing for larger sample volumes. These plates are commonly used in various applications, such as cell culture, assay development, and sample storage.
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Lab products found in correlation
Ff80hp, by GE Healthcare (1 mentions)
Superdex 200 10 300 gl, by GE Healthcare (1 mentions)
A 905, by GE Healthcare (1 mentions)
Megax dh10b t1r electrocomp cells, by Thermo Fisher Scientific (1 mentions)
Xl1 red, by Agilent Technologies (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Galactose, by Merck Group (1 mentions)
Gas permeable microplate adhesive film, by Avantor (1 mentions)
Microplate orbital shaker, by Avantor (1 mentions)
8 protocols using 96 well deep well plate
1
Lateral Flow Detection of iSDA Amplicons
2
Preparative Separation of BSA Conjugates
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Fast protein liquid chromatography (FPLC) was performed for the preparative separation of BSA conjugates. An ÄTKA purifier system equipped with an autosampler A-905 and a Fraction Collector Frac-950 (GE Healthcare, Sweden) was used. The separation of BSA–PEtOx conjugates was performed on a Superdex 200 10/300 GL (GE Healthcare, Uppsala, Sweden) SEC column using a 0.05 M phosphate buffer (pH 7.0) and 0.15 M NaCl solution. The column was loaded with 300 μL of sample and the system was run at a flow rate of 0.5 mL min−1. Fractions of 250 μL were collected into a 96-well deep well plate (VWR, Radnor, PA, USA). A UV detector continuously measured the relative absorbance of the mobile phase at 280 nm. The yields were not optimized.
3
Mutagenesis of Lactam-Biosensing pMGT1 Plasmid
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Mutagenesis of the lactam-biosensing pMGT1 tetA reporter plasmid was performed in vivo in the E. coli mutator strain XL1-Red (Agilent Technologies, Santa Clara, CA). The plasmid was transformed into XL1-Red competent cells via electroporation and plated on LB agar plates supplemented with spectinomycin. After incubating at 37 °C overnight, ~2000 colonies were scraped and were then resuspended in 50 mL of LB medium supplemented with spectinomycin and grown overnight at 37 °C. Afterward, 1 mL of the culture was removed and stored at −80 °C for future plasmid isolation. This overnight was then used to inoculate 500 µL of LB spectinomycin 1:100 in each well of a 96-well deep-well plate (VWR, USA). Passaging of cultures was done in a 96-well format to help prevent selective sweeps by non-mutator phenotypes of XL1-Red that may arise. Passages were performed for five days every 20 hours, by inoculating fresh media in 96-well plates via a pin-replicator. Each day after passaging, culture from all wells was pooled and frozen for subsequent plasmid isolation. Plasmids from all passages were isolated and pooled and then transformed into E. coli MegaX DH10B T1R Electrocomp Cells (Invitrogen, Carlsbad, CA) via electroporation, yielding roughly twelve million transformants. These transformants were pooled and frozen at −80 °C for later use.
4
Recombinant E. coli Protein Expression
5
Yeast Expression of α-Synuclein and Pep4
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Yeast strains were grown in synthetic complete (SC) medium containing 0.17% yeast nitrogen base (Difco, BD Biosciences), 0.5% (NH4)2SO4 and 30 mg/L of all amino acids (except 80 mg/L histidine and 200 mg/L leucine), 30 mg/L adenine and 320 mg/L uracil with 2% D-glucose (SCD) or 2% D-galactose (SCG) for GAL10-driven expression of α-Syn, Pep4WT or Pep4DPM. All media were prepared with double distilled water and subsequently autoclaved (25 min, 121°C, 210 kPa). Amino acid mixtures were sterilized separately as 10× stocks and added after autoclaving. For solid media, 2% agar was admixed. Full media (yeast extract peptone dextrose, YEPD) agar plates contained 1% yeast extract (Bacto, BD Biosciences), 2% peptone (Bacto, BD Biosciences) and 4% D-glucose.
Experiments were performed using overnight cultures grown in SCD for 16–20 h at 28°C and 145 rpm. These cultures were inoculated in 10 mL SCD (in 100 mL Erlenmeyer flasks) to an OD600 of 0.1 and grown to OD600 0.3. Subsequently, cells were transferred into 96-well deep well plates (VWR), whereat 500 μL of cell culture was used per well. After pelleting, cells were shifted to 500 μL SCG per well for induction of expression. For inhibition of Pep4 enzymatic activity, 50 μM pepstatin A dissolved in DMSO (Sigma) were added to cells upon galactose-mediated induction of expression.
Experiments were performed using overnight cultures grown in SCD for 16–20 h at 28°C and 145 rpm. These cultures were inoculated in 10 mL SCD (in 100 mL Erlenmeyer flasks) to an OD600 of 0.1 and grown to OD600 0.3. Subsequently, cells were transferred into 96-well deep well plates (VWR), whereat 500 μL of cell culture was used per well. After pelleting, cells were shifted to 500 μL SCG per well for induction of expression. For inhibition of Pep4 enzymatic activity, 50 μM pepstatin A dissolved in DMSO (Sigma) were added to cells upon galactose-mediated induction of expression.
6
Genome-wide fitness profiling of P. putida
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BarSeq-based experiments utilized the P. putida RB-TnSeq library, JBEI-1, which has been described previously (Thompson et al., 2019a ). An aliquot of JBEI-1 was thawed on ice, diluted into 25 mL of LB medium supplemented with kanamycin and grown to an OD600 of 0.5 at 30 °C. Three 1 mL aliquots of the library were pelleted and stored at −80 °C to later serve as the t0 of gene abundance. Libraries were then washed in MOPS minimal medium and diluted 1:50 in MOPS minimal medium with 10 mM p-coumarate, ferulate, benzoate, p-hydroxybenzoate, protocatechuate, vanillin, vanillate, phenylacetate, or D-glucose. Cells were grown in 600 μL of medium in 96-well deep well plates (VWR). Plates were sealed with a gas-permeable microplate adhesive film (VWR, USA), and then grown at 30 °C in an INFORS HT Multitron (Infors USA Inc.), with shaking at 700 rpm. Two 600-μL samples were combined, pelleted, and stored at −80 °C until analysis by BarSeq, which was performed as previously described (Rand et al., 2017 (link); Wetmore et al., 2015 (link)). All fitness data are publicly available at http://fit.genomics.lbl.gov .
7
Flow Cytometric Analysis of Silencing Loss
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For each CRASH strain, 10 single colonies were inoculated separately into 2 mL of SD-Trp-Leu medium in 96-well deep-well plates (VWR) and were grown overnight to saturation at 30 °C on a microplate orbital shaker (VWR). Overnight cultures were diluted in 1 mL of fresh medium at a density of 105 cells/mL in 96-well deep-well plates and were grown at 30 °C on a microplate orbital shaker until mid-log phase. For each culture, a minimum of 50,000 events were collected using an MACSQuant® flow. Gating was used to include only unbudded and budded cells and to measure separately the number of GFP+ cells and the number of RFP+ cells. A Boolean logic gate was used to determine the number of cells that were both GFP- and RFP-fluorescent. The apparent rate of silencing loss is calculated as the number of cells that are both GFP- and RFP-fluorescent divided by the total number of cells.
8
Hypochlorous Acid and HOSCN Regulation of rclR and rclX
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PA14 WT and ΔrclR strains containing rclR-lacZ and rclX-lacZ plasmids, and clinical CF isolates 1, 2, and 3 containing the rclX-lacZ plasmid, were grown in LB medium with 100 μg/ml tetracycline overnight. Cultures were subcultured 1:50 (OD, 0.05 ± 0.02) in LB medium in 2.2-ml 96-well deep-well plates (VWR) and grown to exponential phase (3 h) and then were left untreated or treated with 2.2 mM HOCl or 0.8 mM HOSCN. Cultures were collected 30 min after treatment with HOCl or HOSCN. β-Galactosidase activity was assayed using the modified version (89 (link)) of the Miller method (90 ).
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