Luria broth medium

Bacteria glycerol stocks of sequenced-verified MISSION^® shRNA lentiviral plasmids (Sigma-Aldrich, St. Louis, MO, USA) targeting tumor suppressors (SH0511) and DNA Repair Pathway (SH1811) were grown in the complex medium Luria Broth Medium (Sigma-Aldrich) with 75 µg/mL of ampicillin and incubated at 37 °C shaking at 200 rpm overnight. Plasmids were isolated with GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich) and used to generate lentiviral virion production.

Bacterial strains used in this study (Supplementary Table S1) were purchased from the Beijing Microbiological Culture Collection Center or provided by the Institute of Clinical Pharmacology, Peking University. All bacterial strains were mixed with 2.5% glycerol and frozen in a cryopreserved tube (Jinzhang, Tianjin, China) at −80°C for storage. For experiments, the frozen strain was thawed and cultured on agar plates with Luria broth (LB) medium (Sigma Aldrich) at 35°C for 24 h before use. Antibiotic solution (gentamicin) was filtered through a 0.22-μm sterile syringe filter (Millipore Millex, Burlington, MA) and stored at −80°C before use.

TAT-fused Uch-L1 was provided by Dr. Ottavio Arancio (Professor at Columbia University), the construct was obtained as described by Gong et al. (2006 (link)).
Briefly, TAT vectors were transformed into E. Coli BL21(DE3) pLysS competent cells (Novagen), and the obtained colonies were grown as 1 ml overnight cultures in Luria broth (LB) medium (Sigma-Aldrich) with 100 mg ampicillin, in the presence of 100 mM IPTG. Then the cultures were transferred to 500 ml LB ampicillin plus 200 mM IPTG to obtain large-scale preparations. Fusion proteins were purified according to ProBond purification system (Invitrogen).
VUch-L1 fusion proteins were i.p. injected into mice at 0.03 g/kg, 20 min before the Rose Bengal injection and surgery procedure. After 6 or 12 h, mice were sacrificed and protein extracts were prepared and examined as described below.

Strains of Acinetobacter baumannii (ATCC #17978) and Staphylococcus aureus (ALC1743) expressing green fluorescent protein (gfp) were used in this study for fluorescence imaging purposes. A. baumannii was provided by Professor Eric P. Skaar of the Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN, and an S. aureus strain was provided by Niles Donegan of the Giesel School of Medicine at Dartmouth College, Hanover, NH. Cultures were grown as per published protocols72 (link)73 (link)74 (link). Briefly, cultures were grown in 20 g/L (1×) Luria Broth (LB) medium (Sigma-Aldrich, catalog # L3522) supplemented with ampicillin (100 μg/mL; Sigma-Aldrich, catalog #A5354) and in 40 g/L (1×) tryptic soy broth (TSB) medium (Fisher Scientific, catalog #211825) supplemented with chloramphenicol (10 μg/mL, catalog #C1919-25G). All cultures were grown overnight at 37 °C at an agitation speed of 70 rpm on a rotary shaker.

The strains used in the study are listed in Additional file 1: Table S1. E. coli DH5α was used for cloning and plasmid propagation, and was grown in Luria Broth (LB) medium (Sigma) supplemented with 50 mg/L kanamycin when necessary; agar (Sigma) was added to 15 g/L for the preparation of medium-agar plates. C. glutamicum ATCC 13032 was used as the host for flavonoid production in this study. C. glutamicum cells were generally grown in Brain Heart Infusion (BHI) medium (BD) and kept in BHI with glycerol (20%, v/v) at − 80 °C for long-term storage. Fermentation by C. glutamicum was conducted in AMM medium supplemented with 0.2 mg/L biotin [62 (link)]. AMM medium contained (per liter): glucose, 20 g; KH₂PO₄, 3.5 g; K₂HPO₄, 5.0 g; (NH₄)₂HPO₄, 3.5 g; casamino acids, 2 g; MgSO₄, 0.12 g; CaCl₂, 11 mg; thiamine HCl, 0.5 mg; MOPS, 8.37 g; Tricine, 0.72 g; FeSO₄·7H₂O, 2.8 mg; NaCl, 2.92 g; NH₄Cl, 0.51 g; MgCl₂ 0.11 g; K₂SO₄ 0.05 g; and micronutrient mix ((NH₄)₆Mo₇O₂₄, 0.4 μg; H₃BO₃, 2.5 μg; CuSO₄, 0.24 μg, MnCl₂, 1.6 μg; and ZnSO₄, 0.28 μg).

A total of nine K. pneumoniae strains were isolated from two elderly patients (>60 years) with urinary tract infections (UTI) at Shanghai Public Health Clinical Center, Shanghai, China. Phage 117 and phage 31 used in this study were kindly provided by the Chen lab (Hualien Hospital, Taiwan). All of the strains were stored at –80 °C in Luria Broth (LB) medium (10 g tryptone, 5 g yeast extract, and 10 g NaCl per liter) (Sigma-Aldrich, St. Louis, MO, USA) with sterile glycerol (25%, vol/vol). The bla_KPC gene was detected by PCR and sequencing with K. pneumoniae carbapenemase (KPC) primers KPC-F (5′-TGTAAGTTACCGCGCTGAGG-3′) and KPC-R (5′-CCAGACGACGGCATAGTCAT-3′) [14 (link)].
Proliferation of phage 117 and phage 31 was performed by using the double-layer agar method [15 ]. To obtain high-titer phage stocks, 5 mL SM buffer (50 mM Tris-Cl, pH 7.5, 99 mM NaCl, 8 mM MgSO₄, 0.01% gelatin, Sigma-Aldrich, St. Louis, MO, USA) was added to a fully lysed plate (90 × 15 mm), and the top agar (5 g/L) overlay was shredded with a sterilized inoculation loop. In order to release phage particles, the mixture was collected in a conical tube, stirred gently for at least 2 h at room temperature (RT) and then centrifuged at 12,000× g for 10 min at 4 °C. The supernatant was filtrated through 0.22 μm syringe filter (Millipore, Billerica, MA, USA), and the phage lysate was titrated and stored at 4 °C.