ApaI restriction enzyme

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The three fragments were first obtained separately by PCR, and then connected by crossover PCR in the following order: SpeI-upstream sequence-downstream sequence-NptI-ApaI. The complete insert DNA strand was digested with SpeI and ApaI and ligated to pDM4.

Protocol tips
The three fragments were first obtained separately by PCR, and then connected by crossover PCR in the following order: SpeI-upstream sequence-downstream sequence-NptI-ApaI. The complete insert DNA strand was digested with SpeI and ApaI and ligated to pDM4.

Publication protocol

The A1S_0316-deleted mutant of A. baumannii was constructed via the markerless gene-editing method using pDM4:A1S_0316. The DNA sequence of the A1S_0316 open reading frame (ORF) was identified from the information of A. baumannii ATCC 17978 in the NCBI database. As the markerless gene-editing method uses the bacterial feature of homologous recombination, the primer sets were designed to result in the overlap of the sequence upstream of A1S_0316 (1004 bp), the sequence downstream of A1S_0316 (887 bp), and the NptI fragment (KmR, used for antibiotic selection, 1.2 kb). SpeI (Enzynomics Inc., Daejeon, Korea) and ApaI (TaKaRa Bio. Inc., Otsu, Japan) restriction enzyme sites were added to the 5′ end of the upstream forward primer and the 3′ end of the NptI reverse primer, respectively. The template used for the upstream and downstream fragments was A. baumannii ATCC 17978. The template used for NptI was pOH4 [12]. The primers used in this experiment are listed in Supplementary Table S2. The three fragments were first obtained separately by PCR, and then connected by crossover PCR in the following order: SpeI-upstream sequence-downstream sequence-NptI-ApaI. The complete insert DNA strand was digested with SpeI and ApaI and ligated to pDM4. The final cloned plasmid was confirmed through sequencing analysis. The confirmed cloned plasmid was then inserted into the E. coli sm10 strain for conjugation. pDM4:A1S_0316 was delivered from E. coli sm10 λ pir cells (donor cells) to A. baumannii ATCC 17978 cells (recipient cells) during the overnight incubation of the bacterial mixture on a fresh LB agar plate at 30 °C. The first selection was performed by collecting the bacterial mix, followed by serial dilution by 10-fold, spreading on a Tri/Kan LB agar plate, and incubation at 37 °C, to select the A. baumannii that contained the plasmid. The colonies collected during the first selection were then spread onto a 10% (wt/vol) sucrose LB agar plate, to select the A. baumannii that had correctly undergone homologous recombination.

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