Ts gelred nucleic acid dye

Tryptone and yeast extract were purchased from OXOID (Shanghai, China), isopropyl β-D-1-thiogalactopyranoside (IPTG, >99%) and kanamycin sulfate (>99%) were bought from Sangon (Shanghai, China) and steroid compounds were purchased from Aladdin (Shanghai, China). All chemicals were of chemical purity and commercially available. Prime STAR Max DNA polymerase was bought from Takara (Shanghai, China), T5 super PCR Mix (Colony) DNA polymerase, DNA Maker, Trelidf™ Prestained Protein Ladder and TS-GelRed Nucleic acid dye were purchased from TSINGKE (Beijing, China). T5 exonuclease was obtained from New England Biolabs (Beverley, MA).

DNA was extracted by using a DNA extraction kit (Ezup Column Bacteria Genomic DNA Purification Kit, B518255-0100, Sangon, Shanghai, China) in accordance with the instructions. DNA concentration and purity were measured by using a NanoDrop2000 microspectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and DNA was analyzed on 1% agarose gel electrophoresis (AGE) containing TS-GelRed nucleic acid dye (0.1 µL/mL; TSJ002, TsingKe, Beijing, China). The extracted DNA was stored at −20 °C.
The extracted genomic DNA was subjected to PCR amplification by using 16S rDNA universal primers [26 (link)] to verify that it qualified for the subsequent experiments. The reaction system had a total volume of 25 µL and comprised 2.5 µL of 10× buffer, 2 µL of the dNTP mixture, 1 µL of each primer (10 µM), 0.2 µL of rTaq DNA polymerase (TaKaRa Biotechnology Co., Ltd., Dalian, China), 2 µL of template DNA, and 16.3 µL of ddH₂O. The amplification procedure was as follows: initial denaturation at 95 °C for 5 min; 30 cycles of denaturation at 95 °C for 30 s, primer annealing at 58 °C for 30 s, and primer extension at 72 °C for 30 s; and final extension at 72 °C for 10 min. All amplification products were analyzed on 2% AGE.

DNA restriction enzymes (BamHI, NotI, and SacI) were purchased from Takara Biomedical Technology Co., Ltd (Beijing, China). The Rapid Yeast Genomic DNA Isolation Kit, Plasmid DNA Mini-preps Kit, SDS-PAGE Preparation Kit, DNA Markers, Protein Markers, DNA Gel Extraction Kit, and the primers for α-factor, 5′-AOX, and 3′-AOX were supplied by Sangon Biotech Co., Ltd (Shanghai, China). TS GelRed Nucleic Acid Dye and T5 Super PCR Mix were obtained from Beijing Tsingke Biotech Co., Ltd (Beijing, China). Hemin (CAS: 16009-13-5, purity: 98%) and biliverdin hydrochloride (CAS: 55482-27-4, purity: 97%) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Geneticin (G418) was purchased from Invitrogen Corporation (Carlsbad, CA, USA). All other reagents were commercially available in analytical and biological grades.