Endotoxin free plasmid extraction kit

Brucella abortus wild-type strain 2308 (S2308) (The Center of Chinese Disease Prevention and Control, Beijing, China) was cultured in tryptic soy broth (TSB) or tryptic soy ager (TSA) (Difco, MI, USA) at 37°C in 5% CO₂ incubator, the Brucella strain was manipulated in a biosafety level 3 laboratory. Escherichia coli DH5α (The Center of Chinese Disease Prevention and Control, Beijing, China) was cultured in Luria-Bertani medium, when appropriate, 100 μg/mL of ampicillin or kanamycin was added to the culture medium. pcDNA3.1 (Youbio,Wuhan, China) and pGL3 plasmid (Promega, Beijing, China), and other constructed plasmids were extracted using Endotoxin-free plasmid extraction kit (TianGen, Beijing, China) for cells transfection.

The ~140 bp substrate pools were synthesized at BGI Shenzhen (Supplementary Data 5). The fragments were amplified, gel purified, and inserted into psiCHECK2 vector by Gibson assembly. The reactions were performed using NEBuilder HiFi DNA Assembly Master Mix (NEB) by mixing the fragments with the linearized vector in a 7:1 molar ratio. Four microliter assembled products were used for bacteria transformation. Bacteria were shaken in SOC medium at 250 rpm for 60 min and then separated on 14 cm LB agar ampicillin selective plates. After 37 °C incubation for about 12 h, eight plates of bacteria were harvested. Plasmids were extracted with the endotoxin-free plasmid extraction kit (TIANGEN).

After overnight growth, the pH and OD600 of the culture medium were measured. The target pH was approximately 7, and the target OD600 was 4–6. Then, 400 ml of the overnight culture was combined with 400 ml of 1 × induction medium (400 ml of fresh LB medium, 16 ml of 1 N sodium hydroxide and 0.4 ml of 20% L-arabinose). The mixture was incubated again at 32 °C and shaken at 250 rpm for 5 h. The total induction time was 5 h. One millilitre of the bacterial culture was used to perform a miniprep, followed by restriction digest analysis to check the quality of the minicircle plasmid. Bacteria were pelleted at 4 °C, and the pellet was stored at − 80 °C.
An endotoxin-free plasmid extraction kit (catalogue number DP117, TIANGEN) was used to extract the plasmid from the bacterial pellet. Then, the plasmid was cut using the restriction site SalI to assess whether the minicircle was successfully induced. The minicircle size was smaller than that of the parent plasmid, and there was no parent plasmid contamination.