Max efficiency stbl2 competent cells

The S. aureus SC01 (MRSA) and SH1000 strains were kindly provided by Dr. Jorge Antonio Benitez’s Laboratory and were grown on Mueller–Hinton agar plates with glucose and without antibiotics at 37°C overnight. The E. coli (Invitrogen MAX Efficiency Stbl2 Competent cells) strains cells were grown overnight at 30°C in LB Broth without antibiotics.

The interaction between the antagonist and DnaK was determined by immune precipitation with anti-Flag M2 Affinity and Western blot analysis. E. coli (Invitrogen MAX Efficiency Stbl2 Competent cells), S. aureus (SH1000), and S. aureus (SC01) were grown in media without antibiotics and then lysed. The lysate was then screened using anti-flag M2 affinity gel and either the SMRwt or SMRmut peptide, or in the absence of both peptides. The eluted protein samples were separated using SDS-PAGE on 4–20% or 8–16% Tris-HCl Criterion precast gels (Bio-Rad) and transferred to the nitrocellulose membrane, 0.45 µm. The membrane was washed in Tris-Buffered Saline (TBS; Bio-Rad) for 5 minutes, blocked with 5% nonfat milk in TTBS (TBS with 0.1% Tween 20) for 1 hour by shaking at room temperature, processed for immunoblotting using a specific primary mouse DnaK antibody by shaking at 4°C overnight, followed by a secondary HRP-conjugated IgG (H + L) antibody. Protein bands were detected using Western Blotting Luminol Reagent (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed by exposure to Image Quant LAS 4000 (FUJIFILM Medical Systems USA, Inc). Densitometry analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD).

The pHRSIN.pSFFV MCS(+) pGK Hygro plasmid (referred from here onwards as pHRSIN) was kindly provided by Paul Lehner. MCherry or 16 copies (16x) of Broccoli was inserted into the multicloning site of the pHRSIN plasmid. The pHRSIN plasmid was digested with BamHI, followed by Klenow (New England Biolabs) treatment and afterwards KpnI and CIP. mCherry and 16xBroccoli plasmids were digested with AfeI and KpnI. Ligation of the correct sequences followed, using T4 ligase and incubation at room temperature. Afterwards, MAX Efficiency Stbl2 competent cells (Invitrogen) were transformed by heat-shock with the ligation reaction and grown in LB agar plates for clone selection. For confirmation, clones with the correct insertions were sequenced before proceeding. Due to the length of all constructs above 16xBroccoli, further cloning with the remaining Broccoli repeats into pHRSIN was not successful. HEK-293T or HeLa cells were transfected with pHRSIN, pHRSIN-mCherry, or pHRSIN-16xBroccoli plus psPAX2 and CMV.VSVg plasmids at a ratio 5:3:1, respectively. Following transfection, we collected the supernatant 48hrs later and transduced new HEK293T or HeLa cells. 48 hours later, cells were split and grown in media supplemented with 150μg/ml hygromycin for positive selection.

The motif-optimized envelope nucleotide sequences were synthesized and cloned into the pUCminusMCS cloning vector by Blue Heron Biotechnology (Bothell, WA). The 2.5 kb envelope fragment was digested with NheI and MluI enzymes (New England Biolabs, Ipswich, MA) and gel extracted from the pUCminusMCS cloning vector before ligation to the SAP-treated pEMC^* expression vector with a Roche rapid DNA ligation kit (Roche Diagnostics, Indianapolis, IN). MAX Efficiency Stbl2-competent cells (Invitrogen, Carlsbad, CA) were transformed and grown at 30°C for 24 h. Clonal populations were screened by colony PCR. Positive colonies were grown in small liquid cultures at 30°C for 24 h and glycerol stocks and plasmid minipreps (Promega, Madison, WI) were generated.

Regions upstream and downstream of TTHA1953 were amplified from the T. thermophilus HB8 genome (isolated using the Quick-DNA Fungal/Bacterial Miniprep kit [Zymo Research]) using the 1953_UpF_HindIII/1953_UpR_KpnI and 1953_DownF_PstI/1953_DownR_AatII primers (File S1), respectively. The thermostable kanamycin resistance (KanR) gene, as well as its accompanying promoter, was amplified from the TTHA1292-disruption plasmid (RIKEN Bioresource Research Center, TDs07G02) using ProKan_F_KpnI/ProKan_R_PstI primers (File S1). The downstream region of TTHA1953 and the KanR fragment was digested with PstI (NEB) and ligated with T4 ligase (NEB). The resulting product was amplified using the ProKan_F_KpnI/1953_DownR_AatII primers and digested with KpnI (NEB). The upstream region of TTHA1953 was also digested with KpnI and ligated to the TTHA1953Downstream/KanR fusion product. This fragment was isolated by gel extraction (Zymoclean Gel DNA Recovery kit [Zymo Research]), digested with HindIII (NEB) and AatII (NEB), and ligated into a pUC19 backbone. MAX Efficiency Stbl2-competent cells (ThermoFisher) were transformed with the resulting ligation reaction, and successful transformations were isolated on kanamycin-containing agar plates. Ultimately, this created a plasmid containing ∼500 bp of genomic sequences found upstream or downstream of TTHA1953, separated by the KanR sequence.

All molecular cloning, of constructs with TANGO1, was carried out using MAX Efficiency Stbl2 Competent Cells (Thermo Fisher Scientific), following manufacturer’s instructions.