Xl10 gold ultracompetent escherichia coli

Gene expression vectors were constructed either by employing restriction endonucleases (New England Biolabs, Ipswich, MA, USA) followed by ligation using T4 DNA ligase (New England Biolabs, cat. no. M0202L) or by Gibson assembly (New England Biolabs, cat. no. E2611L). For restriction enzymes‐based cloning, digested plasmid backbones were dephosphorylated with antarctic phosphatase (New England Biolabs, cat. no. M0289L) before ligation. PCR reactions were performed using either Phusion High‐Fidelity DNA polymerase (New England Biolabs, cat. no. M0530L) or Q5 High‐Fidelity DNA polymerase (New England Biolabs, cat. no. M0491L). For Gibson assembly, the PCR products were amplified using primers that had 15–20 bp complementary sequences at each end. Detailed information about restriction enzymes and primers used for the design of each plasmid is presented in Table S1, Supporting Information. Plasmids were transfected and propagated in XL10‐Gold ultracompetent Escherichia coli (New England Biolabs, cat. no. C2992) and DNA was extracted using a plasmid mini‐prep kit (Zymo Research, Irvine, CA, USA, cat. no. D4054) or a ZymoPURE II Plasmid Midiprep Kit (Zymo Research, cat. no. D4200). A PureLink HiPure Expi plasmid megaprep kit (Thermo Fischer Scientific, Waltham, MA, USA, cat. no. K210008XP) was used for larger‐scale preparation.

Gene expression vectors were constructed either by using restriction enzymes (New England BioLabs, Ipswich, MA, USA) followed by ligation with T4 DNA ligase (New England BioLabs, cat. no. M0202L) or by Gibson assembly (New England BioLabs, cat. no. E2611L). For restriction enzyme-based cloning, digested plasmid backbones were dephosphorylated with Antarctic phosphatase before ligation (New England BioLabs, cat. no. M0289L). PCR reactions were performed using Q5 High-Fidelity DNA polymerase (New England BioLabs, cat. no. M0491L). For Gibson assembly, the PCR products were amplified using primers having 15–20 bp complementary sequences to each end of the linearized vector. Site-directed mutagenesis was performed using a Q5® Site-Directed Mutagenesis Kit (New England BioLabs, cat. no. E0552S). Detailed information about plasmid cloning is presented in Supplementary Tables S1 and S2. Plasmids were transformed and propagated in XL10-Gold® ultra-competent Escherichia coli (New England BioLabs, cat. no. C2992) and extracted using a plasmid mini-prep kit (Zymo Research, Irvine, CA, USA, cat. no. D4054) or a ZymoPURE II Plasmid Midiprep Kit (Zymo Research, cat. no. D4200).