Apramycin sulfate salt

Zein powder (MW 20 kDa),
ethanol (≥99.7%), l-ascorbic acid (Vit C), phosphate-buffered
saline (PBS) 1×, sodium acetate, calcium chloride (CaCl₂), apramycin sulfate salt, trifluoroacetic acid (TFA), and ethanol/hexamethyldisilizane
(36%) were purchased from Sigma-Aldrich and used as received. Low
methoxy pectin powder was purchased from Silva Team. Lecithin (90%)
soybean was purchased from Alfa Aesar. The cell proliferation reagent
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium] (MTS) and the CellTiter-Glo Luminescent viability
assay kit were obtained from Promega. HDFa, fibroblast basal medium
supplemented with Supplement Pack Fibroblast Growth Medium 2, 0.25%
trypsin–EDTA (1×), 2-(4-aminiodinophenyl)-6-idolecarbamide
dihydrochloride (DAPI), and Alexa Fluor 488 Phalloidin were purchased
from Thermo Fisher Scientific. HaCaT cells were purchased from the
Cell Line Service (Heidelberg, Germany).

The minimum inhibitory concentration (MIC) was determined by a modification of the broth dilution technique. Antibiotics were obtained from Sigma-Aldrich (Kanamycin sulfate K1377, Streptomycin sulfate salt S6501, Gentamicin sulfate salt G1264, Neomycin trisulfate salt hydrate N1876, Paromomycin sulfate salt P5057, Hygromycin B H7772, Apramycin sulfate salt A2024). Dilutions of the antibiotic stock were made in sterile distilled water to which equal volume of 2× concentrated LB broth was added. Amount of stock taken was calculated with respect to final diluted volume. Overnight grown culture was added to the broth to achieve 1:100 dilution. After 24-h incubation at 37°C with shaking at 200 rpm, the MIC was inferred as the lowest concentration at which OD₆₀₀ falls below 0.05 with respect to the blank. Eight independently grown replicates were so tested for their MIC.

Bacterial strains and plasmids used in this study are listed in Supplementary Table. 1. Bacterial cultures were initiated from a single clone and were grown at 37 °C in 5 ml of liquid broth low salt (LB_LS, Luria-Bertani formulation) from Duchefa Biochemie under agitation (166 rcf) or on solid LB-agar plates (25 ml) unless indicated otherwise. 30 µg/ml of Apramycin sulfate salt (Sigma–Aldrich) or 10% of crystalized sucrose (Duchefa Biochemie) were used for the selection/counter selection of recombinant/ transformant clones. Four consecutive passages (~64 h) were done in liquid culture to assess the phenotypical stability of the itrA::ISAba13 isolate as described in Supplementary Fig. 5. Eight hours and 16 h cultures were alternated twice and CFUs were plated from the initial bacterial culture and from the final passage. Quantification of height and area were measured using ImageJ. Statistical analyses were performed using unpaired t test with GraphPad Prism 9. All experiments were carried out in biological triplicates unless stated otherwise.