Wizard genomic dna preparation kit

S. cerevisiae CEN.PK113–7D (MATa URA3 TRP1 LEU2 HIS3 MAL2–8c SUC2) was the host strain for all library constructs and grown at 30 °C in yeast extract–peptone–dextrose media, with 200 μg/mL G418 added when appropriate. Yeast transformations were carried out according to the lithium acetate method (35 , 36 (link)). Chemically competent E. coli DH5α (New England Biolabs) was used as a cloning strain and grown at 37 °C in lysogeny broth media with appropriate antibiotics (100 μg/mL carbenicillin or 25 μg/mL kanamycin) and inducer (100 μL of 40 mg/mL 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) was spread and dried on plates for blue/white screening when appropriate.
All Sanger sequencing reactions were performed by Quintara Biosciences. Plasmid isolations were performed with Qiagen Qiaprep kits. Genomic DNA was isolated using the Promega Wizard Genomic DNA Preparation Kit. Gel electrophoresis was carried out using 1% agarose E-Gels according to the manufacturer’s instructions (Invitrogen). BsaI was purchased from New England Biolabs. BbsI was purchased from Thermo Fisher Scientific. High concentration T4 DNA ligase was purchased from Promega. All PCR primers were ordered from Integrated DNA Technologies. All PCRs used Q5 2× Master Mix from New England Biolabs. PCRs were performed on Eppendorf thermocyclers.

Genomic DNA was extracted using the Wizard Genomic DNA preparation kit (Promega, Madison, WI). The staphylococcus protein A (spa) gene was amplified as described [29 (link), 30 (link)]. The detection of the mecA and PVL genes was determined by PCR [31 (link), 32 (link)]. spa types were assigned using Ridom® StaphType software (version2.2.1; Ridom GmbH, $\mathrm{W}\ddot{\stackrel{-}{\mathrm{u}}}\mathrm{r}\mathrm{z}\mathrm{b}\mathrm{u}\mathrm{r}\mathrm{g}$ , Germany). The Based upon Repeat Pattern (BURP) algorithm was used to group spa types into genetic clusters based on their genetic proximity [33 (link)]. A selected number of isolates were subjected to multilocus sequence typing (MLST) as described previously [34 (link)]. The most common, unique, and/or isolates of interest were chosen for MLST (e.g., those which were present in high numbers, low numbers, or unique in some manner including a novel spa type). Sequence types (STs) were assigned using organism specific MLST database (https://pubmlst.org/saureus/). PHYLOViZ software v2.0 was used to analyze Global optimal eBURST of STs and to draw minimum spanning tree and relatedness of STs as described by Francisco et al. [35 (link), 36 (link)]. Positive (USA 300) and negative controls (reaction mixture without DNA template) were used in mecA, PVL, and spa PCR.

Genomic DNA was prepared by the Wizard genomic DNA preparation kit (Promega, Madison, WI, USA) according to the manufacturer’s recommendations. PCR was performed with Takara ExTaq (Takara Bio, Shiga, Japan). Nucleotide sequencing was performed by a commercial DNA sequencing service (Greiner Bio-one, Tokyo, Japan).
bla_MBL genes were detected and identified by PCR and nucleotide sequencing using primers for bla_IMP and bla_VIM [33 (link)]. bla_IMP-1, bla_IMP-10, and bla_IMP-6 are referred as bla_IMP-1 group because the deduced amino acid sequences of bla_IMP-1 differ by single amino acid from those of bla_IMP-10 and bla_IMP-6. Similarly, bla_IMP-11 and bla_IMP-41 are as bla_IMP-11 group because the deduced amino acid sequences differ by single amino acid. Integrase genes specific for class 1, 2, or 3 integrons were detected by PCR using primers by Shibata et al. [33 (link)]. Integron GC arrays were determined by PCR and nucleotide sequencing by primer walking [34 (link)]. According to INTEGRALL nomenclature, novel completely sequenced GCs, namely from a part of 5′-conserved segment (5′CS) to a part of 3′-conserved segment (3’CS), were submitted to INTEGRALL for the attribution of new integron numbers [12 (link)].