Nunc square bioassay dishes

Large BA plates were prepared by pouring blood agar base no.2 (Oxoid) supplemented with sheep blood (Thermo Scientific; 5% final concentration) into Nunc™ Square BioAssay Dishes (245x245x25 mm, Thermo Scientific). The bacterial indicator strains S. pneumoniae TIGR4, S. anginosus 009–1 and S. aureus HG001 were grown overnight in GM17 or TSB, diluted in fresh medium and grown till an optical density at 600 nm (OD₆₀₀) of ~0.25 (S. pneumoniae TIGR4 and S. aureus HG001) or ~0.14 (S. anginosus 009–1). 450 µl of culture was streaked evenly on a BA plate, using a cotton swab that was humidified with phosphate-buffered saline (PBS). Sputum samples were quickly thawed in a 37°C water bath, vortexed and aliquots of ~15 µl were spotted on BA plates with the respective indicator bacteria. These plates were then incubated overnight at 37°C and 5% CO₂. Images were recorded with a G:Box (Syngene, Leusden, the Netherlands), and antimicrobial activity of sputum samples was assayed by measuring the size of growth inhibition zones with ImageJ [30].

A 160 bp Ultramer DNA Oligonucleotides with 20 random nucleotide barcodes sequence (see below) was generated by IDT (Integrated DNA Technologies, Inc, USA). They were inserted into the linearized vector of pRV∆G-4mCherry from step 2 described above using HiFi DNA Assembly (NEB, USA). The HiFi reaction was mixed as described below:
Re-concentrated pRV∆G-4mCherry PCR amplicons (>300 ng/µL): 1 µL; 160 bp Ultramer DNA Oligonucleotides (0.2 uM): 0.9 µL; Nuclease-free water: 8.1 µL; NEBuilder HiFi DNA Assembly Master Mix (E2621L, NEB): 10 µL.
This reaction mix was incubated at 50 °C for 1 hr. After the incubation, we concentrated the mix to >130 ng/µL with GlycoBlue Coprecipitant. After electroporation transformation with Endura Electrocompetent Cells (60242–1, Biosearch Technologies, USA), the cells were plated into Nunc Square BioAssay Dishes (Catalog number: 240835, Thermo Fisher Scientific, USA). After growing for 14 hr at 37 °C, the bacterial colonies were scraped using bacti cell spreaders (60828–688, VWR, USA) for plasmid library purification with NucleoBond Xtra Midi EF kit (740420.10, Takara).