Versatile Cloning Vector Construction

Three series of vectors were generated on the basis of vectors available from the European Molecular Biology Laboratory (pETMBP-1a, pETTRX-1a, and pETGST), Novagen (pET-26b, and pET-28a), MoBiTec (pHT-01, and pHT-43), Invitrogen (pPICZA and pPICZαA), and from the Glockshuber laboratory (pRBI-DsbC) [50] (link). The inserted sequences for pCri-11, 13, and 14 were amplified from pET-15b-SUMO1 [51] (link), pMIS3.0E [52] (link), and pKLSLt [53] (link), respectively. All vectors were prepared for directional cloning in NcoI or NdeI restriction sites at the 5′end and in XhoI at the 3′end. The gene coding for GFP (UniProt code: B6UPG7; 729 bp), including a multiple cloning site (MCS; from pETMBP-1a; 52 bp), was introduced into all vectors. The insert was cloned between the NcoI or NdeI and XhoI restriction sites and was modified to contain an MscI or NheI restriction site immediately after the NcoI and NdeI sites, respectively. Standard cloning techniques were used throughout [54] . Polymerase chain reaction (PCR) primers and DNA modifying enzymes were purchased from Sigma-Aldrich and Thermo-Scientific, respectively. PCR was performed using Phusion high-fidelity DNA polymerase (Thermo-Scientific) according to the manufacturer's instructions and following a standard optimisation step of a thermal gradient in each reaction. For vector preparation, a number of insertions and mutations introduced or eliminated nucleotide sequences. We followed a PCR-based strategy described elsewhere [55] (link), including a DpnI digestion step to remove parental DNA. Digestion with restriction enzymes was carried out according to standard protocols. When necessary, a second round of digestion was performed before the final DNA purification step. DNA was purified from PCR reactions, enzymatic reactions, agarose gel band extractions, and vector extractions using OMEGA-Biotek purification kits. Chemically competent E. coli DH5α, BL21 (DE3), and Origami 2 (DE3) cells (Novagen) were prepared and transformed following Hanahan method [56] (link). Competent cells of P. pastoris KM71H (Invitrogen) and B. subtilis WB800N (MoBiTec) were prepared according to the manufacturer's instructions.