Phusion site directed mutagenesis protocol

For generation of PilM_K26A variants, we followed the Phusion site‐directed mutagenesis protocol (New England Biolabs) using pET28a‐Strep‐PilM and pET28a‐PilMN as templates and primers pilM‐K26A‐for and pilM‐K26A‐rev. Strep‐PilM_K26A, His‐PilM_K26A, and His‐PilM_K26A‐PilN‐Strep were purified as described for the wild‐type variants.

Wnt response elements (WREs) were mutated (mWRE) in a pre-existing plasmid, containing firefly luciferase under the control of aromatase promoter region I.3 and II (pGL3-PII-522 WT) [27 (link)], by use of the Phusion site-directed mutagenesis protocol (New England Biolabs, Frankfurt, Germany). Primers: mWRE1 (forward) 5′-GTGAGTCACTcgcgATTCAATAGACAAACTGATGGAAGGC-3′, mWRE1 (reverse) 5′-TCAGGCCATCTCTAGTGAC-3′; mWRE2 (forward) 5′-cgcgAAAAGTCATTTTGGTCAAAAAGG-3′, mWRE2 (reverse) 5′-cgcgAATAGTAAGTTTCTACAGTAAGAAC-3′; mWRE3 (forward) 5′-TGTTTTGAAAcgcgTTTGGCTTCAAGGGAAGAAGATTG-3′, mWRE3 (reverse) 5′-AAAAGGCAATCTCCCAAC-3′ (lowercase indicates mutated nucleotide positions). All constructs were verified by sequencing.