Clone express 2 one step cloning kit

To achieve the overexpression ppk in S. albulus WG-608, the method was performed as described by our previous study (Pan et al., 2019a (link),b (link)) with some modifications. All primers used in this paper are located in Supplementary Table S1. The DNA fragment encoding ppk (Gene ID: 878853) was chemically synthesized (Aenta, Suzhou, China) with codon optimization. Under the catalysis of CloneExpress II One Step Cloning Kit (Vazyme, Nanjing, China), the obtained DNA fragments were ligated into the vector pIB139, respectively, which was digested by NdeI and EcoRI. Subsequently, ligation product was then transformed into competent E. coli DH5α, and exconjugants were picked out from LB plates containing 50 μg/mL apramycin. After validation by colony PCR using the primer pair C-F/-R and DNA sequencing (Aenta, Suzhou, China), the overexpression vectors pIB139-ppk was obtained.
Finally, the overexpression vector pIB139-ppk was separately transformed into E. coli ET12567 for intergeneric conjugation with S. albulus WG-608. To obtain overexpression strain OE-ppk, the transformants were screened on BTN solid medium supplemented with apramycin and nalidixic acid, and the colonies were verified by PCR using the primer pair P-F/-R.

The antibody heavy- and light-chain V genes (VH/VL) were synthesized (Sino Biological, China) and were amplified and cloned into human IgG1 expression vector by using Clone Express II One Step Cloning Kit (Vazyme, China). The equal amounts of heavy- and light-chain plasmids were transfected into HEK293F cells using EZ Cell Transfection Reagent (Life-iLab Biotech, China) with cells grown to a density of 1 × 10⁶ cells per mL. Following transfection, cells were maintained in CD 293 TGE Medium (ACRO, China) and contained 1/10 CD Feed X supplement (ACRO, China) at 37°C in a humidified 5% CO2 incubator rotated at 120 rpm. After 6 days expression, supernatants were harvested and clarified by centrifugation. Subsequent filtered through 0.22-µm filters and incubated with MabSelect (GE Healthcare, USA) at room temperature for 2 h for antibodies affinity purification. After washing, antibodies were eluted from the MabSelect in chromatography columns using 0.1 M Na-Citrate (pH 3.25) and neutralized with 1M Tris-HCl (pH 8.8). Further purified by centrifugal filtration using Amicon Ultrafilter (Merck Millipore, USA) in PBS and concentrated and stored at −80°C.

All of the point mutations on pUAST-NompC-EGFP plasmid were introduced by site-directed mutagenesis using a CloneExpress II One-step Cloning kit (Vazyme) and confirmed via sequencing of the mutation region. Further experiments were performed the same as outside-out and inside-out patch clamp in the wild-type NompC described in the electrophysiological recording.