Colony pcr and sequencing

E. coli Top10 competent cells were used for plasmid construction and propagation. The uspA2 gene of M. catarrhalis Bc5 (GenBank number: AGH27427.1) was amplified from its genomic DNA by colony-PCR using Phusion High-Fidelity DNA Polymerase (New England Biolabs (NEB), Ipswich, MA, USA). The primers (Table S1) were designed with overhangs complementary to the pASK-IBA2 plasmid (IBA BioTAGnology, Göttingen, Germany) for subsequent annealing during AQUA (advanced quick assembly) cloning [31 (link)]. Plasmid pASK-IBA2 was linearized by PCR, also with Phusion High-Fidelity DNA Polymerase and the respective primers. Upon PCR cleanup, the plasmid template was digested with DpnI (NEB) restriction endonuclease for 30 min at 37 °C in Cutsmart buffer (NEB). Thereafter, DpnI was inactivated by incubation at 80 °C for 10 min. Figure S1 shows a simple scheme illustrating the steps involved in AQUA. Successful cloning was verified by colony PCR and sequencing (Eurofins Genomics, Ebersberg, Germany). Finally, the resulting plasmid pASK-IBA2_UspA2 was propagated in E. coli BL21 (DE3) (NZYTech, Lisbon, Portugal) for protein expression and for use in subsequent experiments. This strain will simply be referred as E. coli UspA2 from now on. E. coli BL21 (DE3) carrying the empty plasmid pASK-IBA2 used as a negative control in experiments will be referred to as E. coli IBA.