Nanodrop 1000 spectrophotometer

Gene blocks of MMP-9_Cat WT and variants Des 3 and Des 4 were purchased from IDT DNA, U.S.A. Primary PCR was carried out to generate the megaprimer, which was used to clone the WT MMP-9 pET28a plasmid using Restriction free Cloning [42 (link)]. Mutagenesis primers for Des1 and Des2 were designed and purchased from Integrated DNA Technologies (IDT DNA, U.S.A.) and used to introduce mutations into the WT MMP-9_cat gene with the transfer PCR protocol (TPCR) [43 (link)]. All primers are summarized in Supplementary Table S1. PCR products were run on a 1% agarose gel to confirm the proper size of the expected PCR product (∼6000 bp). PCR products were DpnI digested to eliminate parental bacterial plasmid DNA. pET28a plasmids encoding MMP-9_Cat WT and variants were transformed into Top 10 chemically competent cells and plated on liquid broth (LB) agar plates supplemented with 50 µg ml⁻¹ antibiotic kanamycin (KAN) and left to incubate at 37°C overnight. Five colonies were picked for each mutant and used to inoculate 5 ml LB media supplemented with 50 µg ml⁻¹ of KAN and the cell cultures were grown at 37 °C overnight. The DNA from the grown cultures was isolated using the Mini prep protocol by Geneaid kit (Geneaid, Taiwan) and its concentration was measured using NanoDrop 1000 spectrophotometer. The correct sequences of the MMP-9 mutants were confirmed by Sanger sequencing (HyLabs).

The primers containing mutation (mutagenesis primers) for Des1 and Des2 were designed and purchased from Integrated DNA Technologies (IDT DNA, USA) and cloned into pET28 plasmids using the Transfer PCR protocol (TPCR) [41 (link)]. Gene blocks of MMP-9_Cat variants Des 3 and Des 4 were purchased from IDT DNA, USA. Primary PCR was carried out to generate the megaprimer, which was used to clone the WT MMP-9 pET28a plasmid using Restriction free Cloning [42 (link)]. All primers are summarized in Supplementary Table 1. PCR products were run on a 1% agarose gel to confirm the proper size of the expected PCR product (~6000 bp). PCR products were DpnI digested to eliminate parental bacterial plasmid DNA. pET28a plasmids encoding MMP-9_Cat WT & variants were transformed into E. coli BL21 (DE3) cells and plated on LB agar plates containing the antibiotic kanamycin (KAN) incubated at 37°C overnight. A single colony was inoculated in 10 mL LB containing kanamycin and grown at 30°C overnight, resulting in several colonies the following day. Five colonies were incubated at 37 °C overnight in 5mL LB media supplemented with 50μg/mL of kanamycin to ensure the desired mutagenesis. DNA was isolated using the Mini prep protocol by Geneaid kit (Geneaid, Taiwan) and measured using NanoDrop 1000 spectrophotometer. We confirmed the correct sequence of the mutants with sanger sequencing (HyLabs).