pSA-HNef-FabV

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	Those colonies transformed with pSA-HP24/HNef/HVif-Bla vectors were selected on LB agar plates containing 269 μM (100 μg mL-1) Ampicillin whereas bacteria transformed with pSA-HP24/HNef/HVif-FabV vectors were selected on LB agar plates containing 1 μM Triclosan. For expression experiments, a single colony from a freshly streaked (18–22 h) LB agar selection plate was inoculated into 10 ml of culture broth containing Ampicillin (269 μM) or Triclosan (1, 5, or 10 μM). The seed culture was grown at 30°C while shaking at 250 rpm until OD600 reached to ~1.

Protocol tips

Those colonies transformed with pSA-HP24/HNef/HVif-Bla vectors were selected on LB agar plates containing 269 μM (100 μg mL-1) Ampicillin whereas bacteria transformed with pSA-HP24/HNef/HVif-FabV vectors were selected on LB agar plates containing 1 μM Triclosan. For expression experiments, a single colony from a freshly streaked (18–22 h) LB agar selection plate was inoculated into 10 ml of culture broth containing Ampicillin (269 μM) or Triclosan (1, 5, or 10 μM). The seed culture was grown at 30°C while shaking at 250 rpm until OD600 reached to ~1.

Publication protocol

Sequencing -confirmed expression vectors were transformed into BL21(DE3) E. coli. Those colonies transformed with pSA-HP24/HNef/HVif-Bla vectors were selected on LB agar plates containing 269 μM (100 μg mL-1) Ampicillin whereas bacteria transformed with pSA-HP24/HNef/HVif-FabV vectors were selected on LB agar plates containing 1 μM Triclosan. For expression experiments, a single colony from a freshly streaked (18–22 h) LB agar selection plate was inoculated into 10 ml of culture broth containing Ampicillin (269 μM) or Triclosan (1, 5, or 10 μM). The seed culture was grown at 30°C while shaking at 250 rpm until OD600 reached to ~1. The seed cultures were centrifuged at 3000 x g for 10 min, re-suspend into fresh culture medium containing Ampicillin, and used to inoculate the main culture at a 1:20 dilution (~0.05 OD600) in a baffled flask. Re-suspension in fresh medium was not carried out for cultures containing Triclosan. The cultures were grown at 30°C while shaking at 250 rpm until OD600 reached to 0.5–0.6. The cultures were then equilibrated to induction temperature (37, 30, or 22°C) and expression was induced with 50 μM of Isopropylthio-β-galactoside (IPTG). Induced cultures were grown for different time lengths (4 h at 37°C, 6 h at 30°C or 12 h at 22°C) before pelleting bacterial cells by centrifuging at 5000 x g for 10 min in pre-weighed centrifuge tubes/ bottles.

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