assays/R569_mTn5_screen_for_lack_of_pyoverdine/protocols/mediums.md

+Name	Purpose	Composition
+Tryptic soy broth (TSB)
+Tryptic soy agar (TSA)	Cultivation of bacteria	15 g/L tryptic soy broth 
+Optional: 1% agar bacteriological (w/v)
+Siderophore medium	Bacterial cultivation for pyoverdine fluorescence determination and iron mobilizing capacity determination	0.5x MS medium 
+10 g/L casamino acids
+0.5% sucrose (w/v) 
+1 g/L TSB
+1 g/L MES
+After autoclaving: 
+2x vitamin solution
+1x artificial root exudate solution
+pH=5.7 (with potassium hydroxide)
+sterile filtration

assays/R569_mTn5_screen_for_lack_of_pyoverdine/protocols/screening_protocol.md

+## Fluorescence-based screening for pyoverdine biosynthesis mutants of R569 
+R569 mTn5 mutants were cultured in 96 well plates at 25°C and 180rpm for five days in 50% TSB medium. Pyoverdine-specific fluorescence of bacterial cultures was captured at λex=395nm/λex=470nm emission. Out of ~2,000 mutants analyzed, the fluorescence-based screening delivered twelve R569 mutants that lacked pyoverdine-specific fluorescence but retained median-like growth behaviour (Thresholds: -6xMAD fluorescence, >-1xMAD Abs600). For validation, cultures were pre-grown for five days in 50% TSB before sub-culturing into fresh siderophore medium and growth for five additional days. Finally, bacterial culture density (Abs600) was determined and pyoverdine-specific fluorescence of bacterial culture supernatants was captured at λex=410nm/λem=500nm. For comparison between genotypes, fluorescence capacity was calculated by dividing the pyoverdine-specific fluorescence through culture density.
+Screening: 1 replicate per mutant
+Validation: 3 replicates per WT/mutant

studies/mTn5_library_establishment/protocols/library_protocol.md

+## Establishment of mini-Tn5 transposon mutant collection in R569 
+
+Mini Tn5-mutant collection of R569 was established similarly with only minor changes as described below. Liquid culture of R569 and E. coli strain BW29427 carrying plasmid pUTmTn5Km2 were grown overnight in no antibiotics or 25 µg/ml Kan and 50 µg/ml DAP at 25 °C or 37 °C, respectively. Conjugation was carried out as described above in “Conjugation of E. coli and R401 and selection for first homologous recombination event”. Conjugation patch was resuspended and serially diluted to obtain single colonies. Individual colonies were picked in 100 µl sterile 50% TSB in 96-well culture plates, sealed and left to grow at 25 °C and 180 rpm for 24 h. Subsequently, 100 µl 50% glycerol were added to each well and plates were frozen until further processing. The outer rows and columns were left uninoculated as to avoid positional effects in the subsequent forward genetic screen. Resuspended mating patches were stocked at -80 °C in 700-µl aliquots using a final concentration of 25% Glycerol. 1:4 dilutions were plated onto 50% TSA plates supplemented with 120 µg/ml Kan, 50 µg/ml Rifampicin and 50 µg/ml Zeocin and incubated at 25 °C for 48h. Individual colonies were inoculated in 100 µl 50% TSB supplemented with the same antibiotics at the same concentrations in well plates and incubated at 25 °C and 180 rpm for 48 h. Then, 100 µl 50% glycerol were added to each culture and plates were frozen at -80 °C.