Fastdigest ncoi

ssODN-mediated modification of the GFP target sequence was verified by Restriction Fragment Length Polymorphism (RFLP) of a newly created silent restriction site. The target sequences were PCR amplified from purified gDNA using KAPA HiFi HS ReadyMix, then amplicons were cleaved with FastDigest NcoI (Thermo Fischer Scientific, Cat. No. FD0573) restriction enzyme in FastDigest Buffer (Thermo Fischer Scientific, Cat. No. B64) and incubation for 30 min at 37 °C. Cleaved amplicons were then resolved by gel electrophoresis, acquired on an AE-9000N E-Graph (Atto) gel documentation system and quantified with the ImageJ software.

Full-length ORF of P5 were amplified from genomic DNA of NTHi 3655 and KR271 using primers containing specific restriction sites (Supplemental Table I). Amplicons were digested with FastDigest NcoI and NdeI (Thermo Fisher Scientific, Waltham, MA) for directional cloning into expression vector pET16b (Novagen) to yield recombinant P5³⁶⁵⁵-pET16b and recombinant P5^KR271-pET16b carrying P5 ORF from NTHi 3655 and KR271, respectively. The recombinant plasmid constructs were subsequently transformed into E. coli DH5α for plasmid propagation. Transformants were selected on LB agar containing ampicillin (100 µg/ml). For P5 expression on the surface of E. coli, P5³⁶⁵⁵-pET16b and P5^KR271-pET16b were transformed into E. coli BL21 (DE3) yielding strain E. coli::ompP5³⁶⁵⁵ and E. coli::ompP5^KR271, respectively, and induced with 1 mM isopropyl β-d-thiogalactoside, as described previously (41 (link)).