Two-milliliter overnight cultures were inoculated by picking single colonies from LB agar plates and incubated for 24 h at 37°C and 700 rpm. Competitors were then mixed and diluted 1:100 in a volumetric 1:1 ratio by passaging 5 µl of each overnight culture into 990 µl LB broth in a 96-deep-well plate (VWR, PA). Initial (time 0 [T0]) and endpoint (time 24 h [T24]) CFU values for each competitor were determined by selective plating on LB agar and LB agar containing 50 mg/liter amoxicillin. Competitions were carried out at 37°C in at least triplicates. Relative fitness (w) was calculated by determining the ratio between each pair of competitors using a Malthusian parameter and the equation w = ln(AT24/AT0)/ln(BT24/BT0) (72 (link)), where A and B are the competing strains.
96 deep well plate
Manufactured by Avantor
The 96 deep-well plates are a type of laboratory equipment used for various applications. They feature a grid of 96 individual wells, each with a deep capacity, designed to hold samples or solutions. The plates are made of durable materials and are suitable for a range of laboratory procedures.
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Lab products found in correlation
Bugbuster master mix, by Merck Group (1 mentions)
Nucleospin plasmid easypure kit, by Macherey-Nagel (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Carbenicillin, by Merck Group (1 mentions)
Anhydrotetracycline atc, by Merck Group (1 mentions)
Multiscreenhts hv, by Merck Group (1 mentions)
Multiskan spectrophotometer, by Thermo Fisher Scientific (1 mentions)
9 protocols using 96 deep well plate
1
Competitive Fitness Assay for Bacterial Strains
2
Recombinant Protein Expression Screening
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Each well of a 96-deep-well plate (VWR) was filled with 500 μL TB. Single clones picked from the agar plate previously streaked with sorted cells were used to inoculate the wells, and the cultures were grown overnight at 30°C shaking at 400 rpm. The following day, a new 96-deep-well plate was filled with 460 μL TB and inoculated with 20 μL of the overnight cultures. After cultivation for 3 h as above, recombinant protein expression was induced by adding 20 μL of IPTG to each well (final concentration 1 mM). Four hours post-induction, the plates were centrifuged (5,000 × g, 10 min, 4°C) and the pellets were resuspended in 25 μL BugBuster Master Mix (Merck) and incubated at room temperature for 20 min. Insoluble fractions were pelleted by centrifugation (5,000 × g, 20 min, 4°C) and the supernatants containing the soluble fractions were diluted in PBS. For the 12 clones showing the highest fluorescence in the soluble protein fraction, the production procedure was repeated in 50 mL TB in 500-mL shake flasks. Plasmids from these cultures were isolated using the NucleoSpin Plasmid EasyPure kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions before sequencing (Microsynth Sequlab, Göttingen, Germany).
3
Iterative Microbial Growth Profiling
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Starting inocula were obtained directly from the ‘source’ soil microbiome solution by inoculating 40 μL into 500 μL culture media prepared as indicated above. For each sample and carbon source, 4 μL of the culture medium was dispensed into fresh media plates containing the different single or pairs of CS in quadruplicate. Bacterial cultures were allowed to grow for 48 hr at 30°C in static broth in 96 deep-well plates (VWR). After 48 hr, each culture was homogenized by pipetting up and down 10 times before transferring 4 μL into 500 μL of fresh media, and cells were allowed to grow again. Cultures were passaged 10 times (~70 generations). OD620 was measured after 48 hr growth. Samples were frozen at −80°C with 40% glycerol.
4
High-throughput Microbial Community Cultivation
5
Tagatose Minimal Media Growth Assay
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Three biological replicates of each isolate were grown overnight in 96-deep-well plates (VWR International, cat #10755-248) in LB media. Saturated cultures were spun down at 2500 × g for 10 min, the supernatant was discarded, and the pellet was resuspended in tagatose minimal media. These suspensions were spun again at 2500 × g for 10 min, the supernatant was discarded, and the pellet was resuspended in tagatose minimal media one last time. This culture was then transferred to 96-well-plates (Costar, Corning™ 3788) containing tagatose minimal media (1:200 dilution) and grown for 40 hours in a plate reader (BioTek Synergy™ H1, Shake Mode: Double Orbital, Orbital Frequency: continuous shake 365 cpm, Interval: 10 min).
6
CRISPR Screening of V. cholerae
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V. cholerae El Tor C6706 CRISPRi strains were grown in Luria–Bertani (LB) Lennox (0.5% NaCl). The CRISPRi screen strains were grown in 1.2 mL of media in 96-deep well plates (VWR) at 37 °C shaking at 900 rpm overnight. CRISPRi gene knockdown was induced with anhydrotetracycline (aTc) (Sigma Aldrich) at 10 nM or 50 nM for cultures in exponential or stationary phase, respectively. Carbenicillin (Sigma Aldrich) was used at 100 µg⋅mL−1 to select for the sgRNA-expressing plasmid.
7
Quantifying T7 promoter strengths
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The strengths of the T7 promoters were quantified in the context of the six gene locations of the complete pathway. This was done by replacing each gene (and RBS) with the mRFP reporter (and RFP008 RBS). The background pathway was derived from library member #14 and when one position is being tested, the other five positions remain the pathway enzymes. The strains were cultured in 500 μl TB in a micro-titer plate (VWR® 96 deep-well plates, cat. No: 82007–292) at 30°C for 4.5 h with shaking at 900 rpm (Multitron HTS, Infors USA Inc., Laurel, MD, USA). When the OD600 of the cultures reached 0.8, 0.2 mM IPTG was added. After an additional growth period of 16 h, flow cytometry was performed.
8
High-Throughput Screening of Mutant Taq Polymerases
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Colonies expressing mutant Taq polymerases were randomly picked, replicated, and then grown overnight at 30°C in 96-deep well plates (VWR) containing 750 μl LB/CAM. Overnight cultures (30 μl) were inoculated into fresh media, induced with anhydrotetracycline at OD600nm of 0.3–0.5, and grown overnight with shaking at 30°C. Cells were collected and used to prepare lysates for direct PCR screening or for affinity protein purification (see below). Cell pellets were re-suspended in 50 μl Tris pH 8 containing 4 mg/ml lysozyme, and incubated at 37°C for 10 min to disrupt cell walls and at 75°C for 15 min to inactivate E. coli protein. Lysates were clarified by centrifugation for 15–30 min at 4000 RPM through a 96-well filter plate (Millipore, Multiscreen HTS, HV).
9
Bacillus Species Growth Assay
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Strains were streaked out on BE Starch Agar (0.3% beef extract, 1% starch) plates and grown for 24 hr at 30°C. Seed cultures were started from several colonies (depending on colony size), inoculated into 3 mL 1x bSAM and grown without shaking at 30°C for 24 hr. Cultures were then transferred to a 96-well plate (Corning Cat. No. 3596), and the optical density (620 nm) of 100 μL was measured (Multiskan Spectrophotometer; Fisher Scientific). Cells were harvested by centrifugation at 3,500 rpm for 15 min, washed twice with 1x phosphate buffered saline, and suspended in fresh 1x bSAM media at a concentration of 5 × 105 CFU/μL. Monocultures or combinatorially assembled communities of the six bacilli species were prepared by inoculating 2 μL from each seed culture into 96-deep-well plates (VWR) containing 500 μL of 1x bSAM, regardless of the number of species. The initial density of cells in the six-member consortia was therefore six times higher than in monocultures. Plates were covered with Aerogel film (VWR) and incubated without shaking at 30°C for another 24 hr. Optical density (620 nm) of the grown cultures was measured as above at the end of the new incubation period.
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