Qiafilter plasmid midi kit

To generate a plasmid-carrying ptxD, the synthetic gene was codon-optimised for the chloroplast of C. reinhardtii (Fig. S1). The gene was designed with two TGG→TGA codon alterations and synthesised by GeneArt (ThermoFisher Scientific). SapI and SphI sites were placed immediately upstream and downstream, respectively, of the coding sequence to allow cloning into chloroplast expression vector pWUCA2 (Young and Purton 2016 (link). See: www.chlamycollection.org for sequence details). Plasmid pWUCA2-ptxD was propagated in E. coli DH5α and extracted using a QIAfilter Plasmid Midi kit (Qiagen, Venlo, The Netherlands). To create a ptxD plasmid for the retro-fitting experiment, the whole gene cassette (i.e. ptxD fused to the psaA-1 promoter/5′UTR and the rbcL 3′UTR) was amplified from pWUCA2-ptxD by PCR such that an MluI site and an MfeI site were located at the upstream and downstream ends, respectively (see Table S3 for primer details). This was then cloned into the MluI and EcoRI sites of plasmid pBa3-AX plasmid (Hallahan et al. 1995 (link)) such that the ptxD cassette was located in a neutral region between psaA-3 and trnL2, as illustrated in Fig. 5a. The resulting plasmid was named as pBa3-AX-ptxD. Finally, plasmid pPO3 was created by cloning the trnW^UCA gene into the MluI site of pBa3-AX-ptxD (Figs. S2 and S3).

Plasmids from UCI38 were extracted using QIAfilter Plasmid Midi Kit (Qiagen, Cat.# 12243). Extracted plasmids were then transformed into other clinical isolates and MGH21∆cas through electroporation. Transformants were selected on LB agar plates supplemented with cefotaxime at the concentration of 10 µg/ml. The extracted plasmid DNA was sequenced, assembled and annotated as described before (Cerqueira et al., 2017 (link)).

The secondary PCR products of the SSH were cloned into a pCR^®2.1-TOPO vector and transformed into TOP10 E.coli competent cells (Invitrogen) following the manufacturer's instructions. About 200 colonies were obtained from each subtraction experiment and 100 randomly picked single colonies were grown overnight in 5 ml of liquid LB medium with the appropriate antibiotic. Plasmid DNA purification was carried out using a QIAfilter Plasmid Midi Kit (Qiagen).

The plasmid Myc-DDK-tagged ORF clone of human FCGR3A, transcript variant 1 (OriGene, USA) was propagated in XL10^®-Gold competent cells (Stratagene) and subsequently purified using QIAfilter plasmid Midi kit (Qiagen). The plasmid was linearized and then CD16 mRNA was generated with the mMESSAGE mMACHINE T7 Ultra kit according to manufacturer’s instructions (Ambion).
5 × 10⁶ CD16− monocytes resuspended in 100 μl of Human Monocyte Nucleofactor Solution (Lonza) were mixed with either 20 μg of purified CD16 mRNA or without mRNA (mock) and electroporated in AMAXA-certified cuvettes using a Nucleofector apparatus (Lonza) using program Y-001. After electroporation, cells were immediately mixed with complete IMDM and transferred to 12-well plates containing the same medium. After 10 hrs, the cells were washed once and used for ADCC assay with BATDA-labeled trastuzumab-coated SKBR3 cells at an E:T ratio of 10:1.

Lentiviral Gene Ontology (LeGO) vectors LeGO-Cer2, LeGO-V2 and LeGO-T2 were generous gifts from Drs. Kristoffer Riecken (né Weber) and Boris Fehse at the University Medical Center Hamburg-Eppendorf. Plasmid replications were performed via transformation of NEB 5-alpha competent E. Coli (New England Biolabs, Ipswich, MA) and DNA purification with the QIAfilter Plasmid Midi Kit (Qiagen, Valencia, CA), according to manufacturer’s instructions.
Lentivirus generation and titer calculation followed previously published protocol38 , scaled up 2.75×. Briefly, 1.375E7 HEK-293T cells were seeded in a T175 flask for an 8-hour transfection, for which 27.5 μg LeGO (Cer2, V2 or T2), 27.5 μg pMDLg/RRE, 13.75 μg rPSV-Rev and 5.50 μg pHCMV-VSV-G plasmids were added. Cells were subsequently filter-removed with 100 μm nylon mesh cell strainer and 0.45 μm PVDF syringe filters. For titer determination, HEK-293 T cells were seeded in 24-well plates at 5E4 cells per well, and transduced for 13 hours with 0.1, 0.5, 1.0, 5.0, 10.0, or 100 μL of viral supernatant. Three wells were transduced per supernatant volume. Percent fluorescent cells were determined three days later with flow cytometry. Calcium phosphate transfection kit was purchased from Sigma-Aldrich.

The ardA sequence was amplified using the primers ardA-fwd and ardA-rev (Supplementary Table S3) and genomic DNA from S. aureus BMB9393 as template. The ardA gene was initially cloned into the p-GEM T easy vector (Promega) and subcloned into the expression vector pCN40 using BamHI and EcoRI restriction sites (Charpentier et al., 2004 (link)). The recombinant plasmid pCN40A (pCN40:P_blaZ-ardA) or empty pCN40 were transformed by electroporation into DC10B competent cells (Monk et al., 2012 (link)). The recombinant plasmids (pCN40A) and pCN40 were obtained (QIAfilter Plasmid Midi Kit; Qiagen), and transformed into RN4220 by electroporation (Monk et al., 2012 (link)), yielding the clones 42P40E (RN4220; empty pCN40) and 42P40A (RN4220; pCN40A). Transformants were confirmed by DNA sequencing and the expression of ardA in 42P40E was detected using real-time qRT-qPCR.