Lysogeny broth medium

Lysogeny broth (LB) medium was purchased from Oxoid Limited (Hampshire, U.K.). Methanol and acetonitrile used for liquid chromatography-mass spectrometry (LC-MS) were high-purity solvents from Concord Technology (Minnesota, U.S.A.). Water used in this work was ultrapure deionized water from Millipore Direct-Q. Chemicals were purchased from Sigma–Aldrich, Geneview, J&K, Amresco or Solarbio. Oligonucleotide primers were synthesized by Tsingke Biological Technology (Beijing, China). DNA plasmid Mini prep, fragment gel extraction and restriction endonucleases clean up kits were from Tiangen (Beijing, China). Talon Cobalt resins were purchased from Clonetech (California, U.S.A.). All protein purification chromatographic experiments were performed on an ‘ÄKTA pure’ or ‘ÄKTA prime plus’ FPLC machine equipped with appropriate columns (GE Healthcare). Protein concentrations were determined according to the method of Bradford [12 (link)] using bovine serum albumin (BSA) as a standard, or using NanoDrop One (Thermo Fisher Scientific). The extinction coefficient for each protein was obtained using the ExPASy ProtParam tool.

Tryptone soya medium (agar, TSA; broth, TSB) and Lysogeny Broth (LB) medium were obtained from Oxoid Ltd. Tetracycline (Tc) and ciprofloxacin (CIP) antibiotics were supplied by Sigma-Aldrich; dimethyl sulfoxide (DMSO) and other analytical grade chemicals were supplied by Fisher Scientific. A minimal basal salts growth medium (BSM) was prepared as described by Rushton et al. [21 (link)] without casamino acids. A 3 : 1 cosmetics grade blend of chloromethylisothiazolinone and methylisothiazolinone (CMIT/MIT), and methylisothiazolinone (MIT) were obtained from Dow Chemical Company, USA; dimethyl dimethylol hydantoin (DMDMH) from Lonza, UK; benzisothiazolinone (BIT) and phenoxyethanol (PH) from Clariant International Ltd, Switzerland. Preservative stock solutions were prepared (in sterile distilled water or DMSO for PH) on the day of use and diluted as required.

The strains and plasmids used in this study are listed in Table S1. All the primers are listed in Table S2. E. coli XL1-Blue MRF’ was used for plasmid construction, and E. coli Nissle1917 was used to construct engineered strain for UA degradation. They were cultured at 37°C in Lysogeny broth (LB medium)(#10855001, Oxoid). Kanamycin (A506636, Sangon Biotech), spectinomycin (A600901, Sangon Biotech), and ampicillin (A610028, Sangon Biotech) were used at 50 μg/mL, 50 μg/mL, and 100 μg/mL, respectively.