Dpni endonuclease

For the generation of point-mutated EGFR variants, the pCMV3-EGFR^wt plasmid (Sino Biological, Eschborn, Germany) was used as template. Briefly, a high fidelity Q5 polymerase (New England Biolabs, Ipswich, MA) was used to amplify the whole plasmid with complementary primer pairs, carrying the desired mutation in the form of mismatches to the original plasmid. PCR conditions were: 94 °C for 2 min, 21 cycles of 94 °C (30 sec), 55 °C (1 min) and 68 °C (30 sec/kb). After three cycles, the oligonucleotides for the introduction of the respective point-mutations were added; primer sequences are listed in supplementary table 1c. Afterwards, the PCR mix was treated with DpnI endonuclease (New England Biolabs) to remove parental DNA. The successful introduction of point-mutations was validated by Sanger Sequencing.

Sodium dodecyl sulfate (SDS), alginate (A7003), phenylmethylsulfonylfluoride (PMSF), imidazole, and dithiothreitol (DTT) were obtained from Sigma Aldrich (St. Louis, MO), whereas cloned pfu DNA polymerase was purchased from Stratagene (La Jolla, CA). DpnI endonuclease, protein A-horseradish peroxidase (HRP), polyvinylidene fluoride, and pET28A(+) expression vector were obtained from New England BioLabs (Ipswich, MA), BD Transduction Laboratories (BD Biosciences, Franklin Lakes, NJ), EMD Millipore (Billerica, MA), and Invitrogen (Waltham, MA), respectively. The QIAprep Spin Plasmid Miniprep Kit and Ni-nitrilotriacetic acid (NTA) agarose beads were purchased from Qiagen (Venlo, Netherlands). The ECL Plus Western blotting detection system RPN 2132 was obtained from Amersham Life Science (GE Healthcare, Cleveland, OH). The oligonucleotide primers were synthesized by Bioneer (Daejeon, Korea) and the analysis facility of Chosun University. Acetylated alginate was purified from Pseudomonas as previously described, using an alginate-overproducer, P. alkylphenolia E1 (pAlgG). Purified acetylated alginate was converted to a Na-form by treating with 50 mM ethylene diamine tetraacetic acid (EDTA), and dialysis using 0.5 M NaCl and double distilled water [41 ].

To mutate the core binding site (5′-GCGTG-3′), two primer pairs were provided as shown in Table 1. KAPA HiFi HotStart (KAPA Biosystems, Wilmington, MA, USA) was used to replicate both plasmid strands with designed primer pairs. The newly constructed plasmids were prepared by digestion of the parental strands by DpnI endonuclease (New England Biolabs, Inc., Ipswich, MA, USA), transformation, and plasmid extraction. Mutated sequences were validated by sequencing.

Point mutations in the pilY1 gene were made using a modified in vitro site-directed mutagenesis protocol (51 (link)). Forward and reverse complementary primers consisting of the nucleotide codon sequence encoding the mutation of interest were used to separately amplify the pMQ30 (for chromosomal mutations) and pMQ72 (ectopic expression) parental plasmids with the gene of interest using high-fidelity Phusion polymerase (NEB). After four cycles of amplification, the products of these reactions were combined and amplified for an additional 18 cycles with additional Phusion polymerase added. The parental plasmid was digested for 4 h using DpnI endonuclease (NEB) at 37°C. Following digestion, the PCR product was transformed into competent E. coli S17 cells and selected on LB with 10 μg/ml gentamicin. Plasmid containing the desired point mutation was isolated and confirmed by sequencing. Introduction of mutations on the chromosome was done by conjugation and counterselection as described above. All chromosomal mutations were verified by PCR amplification and sequencing.