Neb5 alpha competent cells

Stock cultures of Escherichia coli NEB5-alpha competent cells (New England Biolabs; Ipswich, MA, USA) containing the plasmid TA2-Gluc/pColdI^{9 (link)} was grown using a sterile Luria Bertani (LB) broth containing 100 mg mL⁻¹ ampicillin. A Lysis buffer containing 81 mL of 1 M sodium phosphate dibasic and 19 mL of 1 M sodium phosphate monobasic in 900 mL deionized water at pH 7.40 was used for sonication. The cells were sonicated using a micro tip for 20 min on-time; 1 s on–off cycle and purified using immobilized metal affinity chromatography using Ni–NTA resin slurry. The crude mixture was incubated with the resin placed on a rotary mixer for 30 minutes at room temperature. The protein TA2-Gluc was bounded to the Ni–NTA resin and was washed using 10 mL wash buffer containing 50 mM Imidazole twice and passed on a column and the flow-through was collected. The purified protein was eluted with ten (10) aliquots of 1 mL 100 mM Imidazole times and visualized using mini-PROTEAN TGX Gels for SDS-PAGE electrophoresis. After, electrophoresis, pure elution fractions were pooled to give 5–10 mL TA2-Gluc, and excess imidazole was removed by dialysis using a 10 kDa cut-off dialysis cassette. The expressed protein concentration was measured using Pierce 660 protein assay kit according to manufacturer's instructions.

Restriction enzymes, T4 ligase, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, Turbofect, Shrimp Alkaline Phosphatase (SAP), Qubit™ dsDNA HS Assay Kit and penicillin/streptomycin were purchased from Thermo Fischer Scientific. DNA oligonucleotides and the GenElute HP Plasmid Miniprep kit used in plasmid purifications were acquired from Sigma-Aldrich. Q5 High-Fidelity DNA Polymerase, NEB5-alpha competent cells, HiFi Assembly Master Mix were from New England Biolabs Inc. NucleoSpin Gel and the PCR Clean-up kit used to clean up DNA from agarose gels were purchased from Macherey-Nagel. ZymoPURE Plasmid Midiprep Kit was from Zymo Research.