Nebuilder hifi dna assembly reaction

A kanamycin resistance cassette insertion mutation of epsA in V. vulnificus was previously described [31 (link)]. An in-frame deletion mutation of the epsS open-reading frame was constructed by allelic exchange employing the counter-selectable suicide vector pWM91. Approximately 1,000 bp upstream (including the epsS start codon) and 1,000 bp downstream (including the epsS stop codon) were amplified by primer pairs US868/US869 and US870/US871, respectively. pWM91 was linearized with XhoI and the 3 fragments were assembled with the NEBuilder HiFi DNA assembly reaction (New England Biolabs). The deletion vector was transformed into SM10λpir and conjugated into WT and the exeA deletion mutant of V. vulnificus. Cointegrants were selected by growth on rifampicin (50 μg/mL) and ampicillin (100 μg/mL) at 30 °C. Transconjugants were grown in LB for 6 h then plated onto LB containing 6% sucrose for counter-selection. Sucrose resistant colonies were screened for ampicillin sensitivity and epsS deletion mutants were confirmed by PCR with primers US872/US873 and immunoblotting with an anti-EpsS antibody.

The modified Exontrap plasmid is shown in Fig. 5a, and the sequence is provided in Additional file 4: Text S1. The test oligos consisted of 11 bp of the upstream exon and 60 bp of the adjacent intron containing the iSNV to be tested. Additional 22-bp exonic and 19-bp intronic sequences homologous to the vector were also included for the seamless insertion of the oligos into the plasmid body. Further, a single nucleotide barcode was introduced to indicate whether the transcript came from the wild-type or variant construct. The 113-bp oligos containing different test iSNVs were synthesized in parallel as a pool using OligoMix (LC Sciences, Houston, TX). In the present study, the ASSET-seq assays contained 82 pairs of reference and variant test sequences. The synthesized oligos were then cleaved from the chip and amplified via high-fidelity PCR with primers paired to the exon and intron homology sequences (Additional file 4: Text S1). The pooled oligos were directionally inserted into the Exontrap plasmid using the NEBuilder HiFi DNA Assembly Reaction (New England Biolabs, Ipswich MA). The assembled plasmids were transformed into bacteria and plated on LB agar plates containing ampicillin. The resulting colonies were scraped and grown in LB + ampicillin medium. Plasmid DNA was isolated using HiSpeed Plasmid Maxi kit (Qiagen, Germantown, MD).