Nebuilder hifi dna assembly protocol

Sequences representing the candidate ORFs were ordered as custom designed double stranded DNA fragments (Integrated DNA Technologies, Belgium). Sequence predicted to encode transmembrane domains was removed and synthetic genes were codon optimised for Spodoptera frugiperda (Sf9) or E. coli to maximise expression in the relevant host. All DNA fragments were flanked with 18 base pairs of sequence homologous to the vector pTriEx 1.1 (EMD Biosciences), suitable for expression in E. coli under control of the T7 promoter or insect cells under control of the baculovirus very late P10 promoter. All clones were generated by Gibson assembly (Gibson et al., 2009 (link)). The sequence designs were such that translation initiated at the bTB ORF initiation codon and fused the C-terminus with the histidine tag present in the vector, except when otherwise indicated. The pTriEx-1.1 cloning vector was linearised by digestion with restriction enzymes XhoI and NcoI (Thermo Fisher Scientific, Paisley, United Kingdom) and 20 ng gene fragment was assembled into 50 ng linearized pTriEx-1.1 cloning vector using the NEBuilder HiFi DNA Assembly Protocol (NEB, United Kingdom). Clones were isolated following transformation of Stellar competent cells (Takara Bio, France) with confirmation by colony PCR followed by DNA sequencing.

This plasmid was created using NEBuilder HiFi DNA assembly protocol (NEB). In short, backbone pAc-GFP-C1-Caprin-1^Δ360–383 was created via Phusion PCR protocol (see above) with primers (electronic supplementary material, table S4) containing a 20 nt overhang to the USP10^1–25 insert. This USP10^1–25 insert was also amplified with primers (electronic supplementary material, table S4) containing a 20 nt overhang to the pAc-GFP-C1-Caprin-1^Δ360–383 backbone via a Phusion PCR protocol (see above) based on a pEGFP-C1-USP10^1–40-WT template [31 (link)]. Both PCR products were mixed with NEBuilder HiFi DNA assembly master mix according to the manufacturer instructions and five alpha E. coli (NEB) were chemically transformed. Plasmids were isolated and sequenced.

The PAO1 lasA mutant (PW4282 lasA-H03∷ISlacZ/hah) was from the PAO1 knockout library [55 (link)]. PA14 deletion mutants were constructed by singly or sequentially deleting the coding sequences of rhlA, pqsL, phzS, and hcnC. Briefly, flanking primers were designed to anneal 800–1,200 bp upstream and downstream of the coding region. The resulting PCR product was inserted into plasmid pEX18Gm in accordance with the NEBuilder HiFi DNA Assembly protocol (New England Biolabs). Mutant alleles were integrated onto the chromosome of PA14 as described previously [56 (link)]. Briefly, pEX18Gm containing the in-frame deletion and gene-specific flanking regions was mated into P. aeruginosa via Escherichia coli S17-λpir. Primary integrants were selected for with gentamycin and irgasan, then grown for 4 h in LB without selection to allow for recombination. Dilutions of P. aeruginosa were plated on LB containing 8% sucrose for counterselection (loss of plasmid). Deletion strains were confirmed through PCR and sequencing (Genewiz). Plasmid PpflB∷gfp was constructed as follows, 298 bp upstream of the pflB coding region was amplified from HG003 genomic DNA using primers flanked with EcoRI and XbaI sites and cloned upstream of gfpuvr in plasmid pALC1434 [57 (link)].