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4 protocols using gentamicin gm

1

GFP-tagged Bacterial Strains in Quorum Sensing Assays

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Bacterial strains tagged with green fluorescent protein (GFP) were used in QS assays with P. aeruginosa PAO1. The lasB::gfp (ASV) [29 (link)], rhlA::gfp (ASV) [30 (link)] and pqsA::gfp (ASV) with GmR [31 (link)] translational reporter fusions were used. Reporter strains were routinely grown overnight in Mueller Hinton Broth (MHB, Oxoid, Thermo Fisher Scientific, VIC, Australia) with 125.6 μM gentamicin (Gm, Sigma-Aldrich, NSW, Australia) at 37 °C with shaking at 180 rpm. For QS assays, reporter strains were grown in ABTGC medium, which is AB minimal medium [32 (link)] plus 7.4 µM thiamine, 0.01 M glucose and 0.01 M casamino acids [33 (link)]. AB minimal medium consists of 15.1 mM ammonium sulfate, 33.7 mM sodium phosphate dibasic, 22 mM potassium dihydrogen phosphate, 50 mM sodium chloride, 1 mM magnesium chloride hexahydrate, 100 µM calcium chloride dehydrate and 1 µM iron (III) chloride hexahydrate [33 (link),34 (link)]. All the chemicals to prepare ABTGC medium were purchased from Sigma-Aldrich, NSW, Australia. For the other experiments in this study, a wild-type P. aeruginosa PAO1 [33 (link)] was grown in MHB overnight at 37 °C with shaking at 180 rpm.
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2

Anaerobic Bacteroides and E. coli Culturing

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Detailed information of the strains used in this study is provided in Supplementary Data 4. All anaerobes were cultured in an anaerobic chamber with an atmosphere of 83% N2, 2% H2 and 15% CO2 at 37 °C. For most experiments, Bacteroides strains, AC, CC were grown in ABB media, ER was grown in ABB media with the addition of 3.3 mM sodium acetate (Sigma) and RI was grown in Brain Heart Infusion Broth (BHI, Sigma). We used E. coli pir2 (Invitrogen) for cloning and maintenance of plasmids with R6K origin (pNBU2-ermGb derivatives and pFW1000 derivatives, Supplementary Data 4). E. coli DH5α (Thermo Fisher Scientific) was used for cloning and maintenance of plasmids with p15A, pSC101ts and ColE1 origins (pFW2000 derivatives, pFW3000 and pFW4000). We used E. coli BW29427 (E. coli Genetic Stock Center, CGSC) for E. coli-Bacteroides conjugations. All E. coli strains were grown aerobically in Luria Bertoni (LB, Sigma) media. To support the growth of E. coli BW29427, we supplemented LB media with 25 μM of 2,6-Diaminopimelic acid (DAP, Sigma). We used the following antibiotics when required including 100 μg mL−1 carbenicillin (Carb, IBI Scientific), 25 μg mL−1 erythromycin (Erm, Sigma) and 200 μg mL−1 gentamicin (Gm, Sigma). We used 1 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG, Gold Biotechnology) for the induction of FnCpf1.
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3

Immune Response Quantification Protocol

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GL was purchased from Sigma-Aldrich (purity ≥95.0%, St. Louis, MO, USA). Gentamicin (GM) was purchased from Sigma-Aldrich (St. Louis, MO, USA). ELISA kits for IFN-γ, IL-12p70, TNF-α, IL-6, and IL-10 were obtained from eBioscience (San Diego, CA, USA). Caspase-1 activity assay kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China).
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4

Bacterial Strains and Plasmids Protocol

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Bacterial strains and plasmids used in this study are listed in Table S1. P. laumondii [28] , E. coli XLI-blue MRF' (Stratagene), WM3064 [29] and MG1655 [30] were routinely grown in Luria-Bertani (LB) broth (Difco or Sigma) or on agar (Difco), nutrient agar (NA) (Difco) as previously described [27] . When required, chloramphenicol (Cm) (Sigma) was added to bacterial cultures to a final concentration of 8 mg/l for P. laumondii mutant strains and 20 mg/l for E. coli strains harboring pJQ200SK-derived plasmids. The final concentrations of gentamicin (Gm) (Sigma) were 20 or 30 mg/l for E. coli and 20 mg/l for P. laumondii strains harboring pJQ200SK derived plasmids. All strains harboring pBBR1-MCS5-derived plasmids were grown in/on LB broth/agar in the presence of Gm at a final concentration of 15 mg/l.
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