Clonexpress 2 multis one step cloning kit

VNP20009 (abbreviated VNP), VNP-RFP (transformed with a plasmid expressing RFP with a J23100 promoter), VNP-LuxCDABE (genomic insertion of LuxCDABE with a J23100 promoter), VNP-PD1nb (transformed with a plasmid expressing PD1nb with a J23100 promoter; a flag tag was added to the N-terminus of PD1nb to facilitate subsequent detection, pJ23100-flag-PD1nb), VNP-NC (transformed with an empty plasmid) and VNP-psifB-RFP (transformed with a plasmid expressing RFP with a sifB promoter; an HA tag was added to the C-terminus of RFP to facilitate subsequent detection, psifB-RFP-HA) were preserved in our laboratory. DH5α and BL21 strains were purchased from Vazyme. All plasmids were constructed using the ClonExpress II/MultiS One Step Cloning Kit (Vazyme, C112/C113, Nanjing, China). The plasmid Pet28a-PD1nb was kindly provided by Dr. Shufeng Li (Southeast University). The construction and screening of anti-PD1 nanobodies are described in Section 2.15. The VNP strains were electrotransformed with the plasmids as described previously³¹ .

Isopropyl-β-d-thiogalactopyranoside (IPTG) was supplied by Sinopharm Chemical Reagent (Tianjin, China). Chloramphenicol, kanamycin, and rifampicin were supplied by Solarbio Science and Technology (Beijing, China). Taq DNA polymerase and Phusion high-fidelity DNA polymerase were obtained from Thermo Fisher Scientific Inc. (MA, USA). ClonExpress II/MultiS One Step Cloning Kit was purchased from Vazyme Biotech (Nanjing, China). Wizard Genomic DNA Purification Kit was obtained from Promega Corporation (WI, USA). Restriction endonuclease BsaI, T4 PNK, and T4 DNA ligase were purchased from New England Biolabs Inc. (MA, USA). All other chemicals used were of analytical grade or better.

VNP20009 (VNP) and VNP20009 expressed IFNβ with J23100 promoter initiation (VNP- IFNβ) were cultured in Luria Bertani (LB) broth or on LB agar plates at 37°C. bacteria growth curve was measured by the microplate reader constantly. All plasmids were constructed by using the ClonExpress II/MultiS One Step Cloning Kit (C112/C113, Vazyme). The morphogens of bacteria were detected by scanning electron microscopy (SEM). When OD₆₀₀ reached 0.6–0.8 (logarithmic phase), measured the expression of the protein in bacteria by sonication lysis bacteria.