Casamino acids

Anoxybacillus caldiproteolyticus 1A02591 (MCCC1A02591) was obtained from Marine Culture Collection of China (MCCC), which was isolated from the deep-sea sediment collected from the hydrothermal vent in the east of the Pacific Ocean (12.71°N, 103.91°W). Casein and elastin were purchased from Sigma (United States). Insoluble type I collagen fiber (bovine achilles tendon) was purchased from Worthington Biochemical Co. (United States), gelatin from Boston Biomedical Inc. (United States), and casamino acids from Sangon Biotech Co., Ltd. (Shanghai, China).

Overnight cultures were adjusted to an OD₆₀₀ of 1.0 and diluted by 1:100 into fresh agrobacterium minimal medium containing 25 μg. ml^-1 thiamine (APExBIO, USA) (ABT) (79 (link)) supplemented with 5 g · L⁻¹ glucose (Sangon Biotech, PRC) and 2 g · L⁻¹ Casamino Acids (Sangon Biotech). Bacterial strains were grown in ABTCG medium with an initial inoculum of OD₆₀₀ of 0.01. An aliquot (100 μL) was transferred to a polystyrene, black 96-well plate (Corning, USA). Arbitrary fluorescence intensity units (FIUs) were measured with an excitation wavelength of 488 nm, and the cell density (OD₆₀₀ values) of different samples was simultaneously measured. Relative fluorescence units (RFUs) were determined by normalizing the FIU values against OD₆₀₀ values as reported previously (78 (link)). Three biological repeats were performed, and each biological repeat contained at least four technical replicates.

SM9913 was originally isolated from a deep-sea sediment (Chen et al., 2003 (link)). Bovine casein was purchased from Sigma-Aldrich chemical Co. (US), and casamino acids from Sangon Biotech (Shanghai) Co., Ltd. (China). Dipeptides and tripeptides were synthesized by Shanghai Jiepi Biotechnology Co., Ltd. (China). The casein medium (pH 7.5) contained 0.5% (w/v) casein, 1% (w/v) mannitol and artificial seawater. The casamino acids medium (pH 7.5) contained 0.5% (w/v) casamino acids, 1% (w/v) mannitol and artificial seawater.