To express ZapE and K72A, the expression vector pET28a was used. The zapE and zapEK72A each were amplified from PAO1 genomic DNA and pUCP20‐K72A with 28a‐zapE‐F and 28a‐zapE‐R. For PqsH, the expression vector pMAL‐C5x was chosen, and the pqsH was amplified with MAL‐pqsH‐F and MAL‐pqsH‐R. Gibson Assembly Master Mix (NEB) was applied to fuse the amplicons with the expression vectors. The fusion gene constructs were then transformed into E. coli strain BL21 (DE3). HIS‐ZapE and HIS‐K72A were purified on Ni NTA beads (Smart Lifesciences) independently
19 (link), and MBP‐PqsH was purified on Dextrin Beads 6FF (Smart Lifesciences) as previously reported
13 (link). The fusion proteins were further purified by ion exchange on a Mono Q 5/50 GL column (GE Biosciences) and then followed by a gel filtration on a HiLoad 16/60 Superdex 200 size exclusion column (GE Biosciences).
19 (link), and MBP‐PqsH was purified on Dextrin Beads 6FF (Smart Lifesciences) as previously reported
13 (link). The fusion proteins were further purified by ion exchange on a Mono Q 5/50 GL column (GE Biosciences) and then followed by a gel filtration on a HiLoad 16/60 Superdex 200 size exclusion column (GE Biosciences).
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