Omnitray

The 30 ml of 1/10 Muller-Hinton broth (MHB) was supplemented with 1% agarose and 5 × 10⁵ CFU/ml bacterial cells was poured into a single-well Omnitray (Nunc) and overlaid with a TSP 96-well plate. Twenty micrograms of each synthesized Chain201D was tested against various strains of bacteria and plate was incubated overnight at 37 °C and the zone of inhibition were recorded. For thermal stability evaluation peptides were diluted to the concentration of 2 mg/ml in PBS buffer (pH 7.4) and incubated at various temperatures, + 4 °C, 25 °C (RT), 37 °C and 45 °C, while the peptides stored at −20 °C were used as control. After each time interval, 100 µl (2 mg/ml) of peptides were taken and stored at −20 °C, until RDA was performed to determine the antibacterial activity. For pH sensitivity evaluation, the peptides were diluted in different pH (pH 4, 5, 6, 7, 8 & 9) and the antibacterial activity of peptides was tested by RDA.

The yeast deletion collections were obtained as individual mutants in 96-well plates that had been stored at −80°C. Both the HIP deletion pool and HOP deletion pool were created separately as previously described [84 (link), 93 ]. The 96-well plates of mutant stocks were thawed completely, after which a 96-well transfer pin was used to transfer a small volume of mutants to a Nunc Omni Tray containing YPD agar with geneticin antibiotic. Between transfers, the 96-well transfer pin was sterilized with ethanol and flamed three times. After growing the cells for 48 h at 30°C, the missing and slow growing mutants were recorded and two times the cell mass of these mutants were added separately. Working in a sterile hood, each tray was flooded with ~10 mL of YPD broth and all formed colonies were gently scraped by sterile cell spreader. The resuspended colonies were transferred into a sterile 1000 mL flask with a sterile stir bar. The suspension was mixed for 5 min on a stir plate to obtain an homogenized pool. The concentration of the freshly prepared pool was adjusted to 125–250 cells/mutant/μL by centrifugation at 500 × g. Once the concentration was adjusted, sterile glycerol was added to 15% (vol/vol) and 200 μL aliquots of the pool were stored in PCR strip tubes at −80°C.

Exponential phase cultures were grown to an OD of 0.3 and plated onto Iso-Grid membranes (Neogen) on supplemented M9 with 1.5% agar at a volume of 0.5 μl per square. Gradients were created by cutting holes in supplemented M9-agar (1.5%) plates (cast in OmniTray [Nunc]) containing 10 μM IPTG. Holes were cut on both ends of a domain to be inoculated at a size of 25% of the domain, each. Holes were then filled with liquid M9-agar to which either 3O-C6- or 3O-C12-HSL had been added at 4X concentration. After hardening, excess agar was cut away leaving each domain isolated. For relay circuit assays, circular holes were punched in the centre of plates using the back of a pipette tip and the holes were filled with ~200 μl of liquid agar containing 40 μM of the appropriate HSL. Plates were sealed with parafilm and imaged using a motorized Leica M205 FA fluorescence stereo microscope controlled using Leica LAS X software. Plates were incubated at 37 °C using a DigiTherm microscope temperature control air bath (Tritech Research). Illumination was an LED white light source (Lumencor) with excitation filters of 426–446 nm, 490–510 nm, and 555–589 nm, and emission filters of 460–500 nm, 520–550 nm, and 608–682 nm. Tiled images were taken every 10 min and were stitched using Leica LAS X software.

The entire GEM prep was washed 3× in 1% BSA in PBS, transferred into manipulation media (CO₂ independent medium, Life Technologies; 10% FBS (Hyclone), 1× Glutamax, 1× Pen/Strep; all from Life Technologies) and dispensed into a single 128 × 86 mm OmniTray (Nunc) tissue culture dish and scanned by eye at 50× magnification on a Leica DMI 6000 fluorescence microscope using a B/G/R triple filter (Chroma). GEMs containing profiles of potential interest were examined at 100× or 200× magnification to confirm profiles. GEMs featuring the desired reporter profile were harvested directly from the dish using a hand-held micropipettor and placed into a well of a 96-well plate containing 6 M guanidium thiocyanate lysis buffer, which rapidly dissolves the GEM matrix, lyses the secreting lymphocytes and preserves the cellular mRNA.

To estimate the level of sensitivity to zeocin in individual tests, we performed a semi-quantitative drop-assay. Each deletion strain was grown in YPD medium with shaking at 28°C to a density of approximately 1–2x10⁷cells per ml. The cells were then spun down, washed with 0.9% NaCl (POCh), and adjusted to a density of 3.3x10⁷ cells per ml via resuspension in the same solution. Five 6-fold serial dilutions in a 0.9% NaCl solution were performed for each strain, and 3.3 μl of each dilution was spotted onto Omnitray (Nunc, Roskilde, Denmark) plates containing selection medium (YPD with 2.5 μg per ml zeocin; 5 μg per ml zeocin; 5 μg per ml zeocin and 50 mM KCl (POCh); or 50 mM KCl alone), or YPD medium for the dilution control. The plates were then incubated at 28°C for 2 days. The countable spots of the colonies grown on all types of plates were totaled, and survival on zeocin was calculated, taking the dilution factor into account.