Escherichia coli xl10 gold

Wild type S. pombe 972h⁻, and auxotrophic ED666 (ade6-M210, ura4-D18, leu1-32) as parental strain were used in this study. Cells (1 × 10⁶ mL⁻¹) were grown in liquid Edinburgh Minimal Medium (EMM2) at 30°C with orbital shaking at 180 rpm. Medium was prepared with 0.67% yeast nitrogen base w/o amino acids, 0.5% or 2% glucose, and supplemented with adenine, histidine, leucine, lysine, and uracil at 50 µg/mL concentration (Petersen and Russell 2016 ). After transformation of S. pombe ED666 with plasmid containing uracil (Ura4⁺) gene as selection marker, selection media contained 2% glucose, and supplemented with only 50 µg/mL adenine and leucine. Plasmids used in this study were constructed, selected, and amplified by using Escherichia coli XL10-Gold (Agilent) cells. E. coli cells were grown in LB medium supplemented with 100 μg/mL ampicillin. All solid media contained 2% agar.

DNA cloning was performed using chemically competent Escherichia coli XL-10 Gold (Agilent Technologies) grown in Luria-Bertani (LB) medium (5 g L^-1 yeast extract, 10 g L^-1 peptone and 10 g L^-1 NaCl, pH 7.2). When needed, agar was added to a final concentration of 1.5%. When zeocin (25 μg mL^-1) was used for bacterial antibiotic selection, the NaCl concentration was reduced to 5 g L^-1.
K. phaffii strains were derived from X-33 (Invitrogen). The LA2 strain (amd2 ade2) was described in previous work [27 (link)]. Yeast was routinely grown in YPD medium (10 g L^-1 yeast extract, 20 g L^-1 peptone and 20 g L^-1 glucose). Solid medium used 2% agar. Zeocin and geneticin, when used, were added at 100 μg mL^-1 and 500 μg mL^-1, respectively. Hygromycin B was used to a final concentration of 50 μg mL^-1. MD medium used 0.34% Yeast Nitrogen Base, 1% (NH₄)₂SO₄, 2% glucose, 0.00004% biotin, and 0.0002% adenine or 0.004% histidine, when needed.

Codon-optimized synthetic sequences were generated by gene synthesis (Synbio Technologies, Monmouth Junction, NJ, USA) and subcloned into a mammalian expression vector pIF (Figure S2). This vector was designed for in vivo DNA delivery and had a reduced backbone size (3.1 kb) with a minimal set of bacteria-originated elements, including the pBR322 origin of replication, kanamycin resistance gene, CMV enhancer/promoter without intron A, and bGH poly(A) signal. Transfection-grade plasmid DNA was isolated from an Escherichia coli XL10 Gold (#200314, Agilent, Santa Clara, CA, USA) cell overnight culture with the NucleoBond™ Xtra Midi Kit (Macherey-Nagel, Düren, Germany).

Putative PLM resistance genes identified in this study were introduced into a PLM-susceptible S. aureus host to assess their ability to confer resistance. DNA fragments corresponding to these genes were either generated by PCR-amplification with Phusion® High-Fidelity DNA Polymerase (NEB) using oligonucleotide primers described in Supplementary Table S1 or were obtained by synthesis (Genewiz). PCR amplicons and synthesized DNA fragments were digested with KpnI and EcoRI (NEB) to enable directional ligation into similarly-digested expression plasmid pRMC2 (24 (link)) for transformation of Escherichia coli XL10-Gold (Agilent Technologies). DNA-sequence verified constructs were then introduced into S. aureus RN4220 (25 (link)) by electroporation (12 (link)). Transformants were grown in cation-adjusted MHB at 37°C with vigorous aeration to an OD₆₂₅ of 0.6, and expression induced with anhydrotetracycline hydrochloride (ATc) (Sigma-Aldrich) at a final concentration of 100 ng/ml for 3 h. Susceptibility testing of these induced cultures was carried out as above, using MHB supplemented with ATc (100 ng/ml).

Chryseobacterium indologenes #3362 was previously isolated from environmental samples obtained from a residential aged care facility as part of a project on the surveillance of the development and dissemination of AMR in residential aged care facilities. Klebsiella pneumoniae D53 is a clinical isolate confirmed to carry NDM-4 and was a kind gift from Ms Jan Bell from AGAR (Australian Group for Antimicrobial Resistance). Escherichia coli XL10-Gold (Agilent) was used for the creation of constructs and plasmid propagation. E. coli BL21(DE3) was used for protein expression and purification. E. coli C41(DE3) cells^{59 (link)} were transformed with pET41a(+) carrying different constructs and used for protein localisation assays and minimum inhibitory concentration (MIC) determinations. Unless otherwise stated, all strains were grown aerobically at 37 °C in Difco^TM Luria-Bertani broth, Miller (BD) supplemented with kanamycin 25 μg/mL. Antibiotics were purchased from Glentham Life Sciences and Sigma-Aldrich. Chemical reagents and oligonucleotides were purchased from Sigma-Aldrich. Enzymes used in cloning were purchased from New England Biolabs.

All transformations for cloning or plasmid amplification were performed in Escherichia coli XL10 Gold (Agilent) according to manufacturer’s instructions.
Viral RNA (vRNA) was extracted from egg-grown viral stocks using QIAamp Viral RNA Mini Kit (Qiagen, 50952904) according to manufacturer’s instructions. From purified vRNA, NA cDNA was produced using NZY M-MulV First-Strand cDNA Synthesis Kit (NZYTech, MB17302) with primer “NA_Fw_HindIII” following manufacturer’s recommendations. To produce pEGFP-N1::NA, NA was then amplified and cloned in HindIII-KpnI restriction sites of pEGFP-N1. To generate pLEX-MCS-1::NA-GFP, NA-GFP was amplified from pEGFP-N1::NA and cloned into NotI/XhoI sites of pLEX-MCS-1. pDual::seg6-E229A and pEGFP-N1::NA-E229A were generated by site directed mutagenesis of pDual::seg6 and pEGFP-N1::NA respectively, using the QuikChange Site-Directed Mutagenesis Kit (Agilent, 200518), according to manufacturer’s instructions. Primer sequences are indicated in S3 Table.

Escherichia coli XL10-Gold used for gene cloning and phagemid amplification was purchased from Agilent (La Jolla, CA, USA). The E. coli TG-1 strain used for phage production and Fab display was obtained from GE Healthcare (Tokyo, Japan). E. coli SHuffle T7 express and restriction enzymes used in this study were purchased from New England Biolabs (Ipswich, MA, USA), while the modification enzymes were purchased from Toyobo Biochemical (Osaka, Japan). The phage display library Tomlinson I and helper phage KM13 were obtained from Source BioScience, Nottingham, UK. SARS-CoV-2 S-pseudotyped human immunodeficiency virus PSV001 and the S protein fragments were obtained from Sino Bio Inc., Beijing, China, unless otherwise indicated. Other chemicals, reagents, and antibodies, unless otherwise indicated, were obtained from Sigma-Aldrich (St. Louis, MO, USA), Fujifilm Wako Pure Chemicals (Osaka, Japan), or Beijing Solarbio Science & Technology Co. Ltd. (Beijing, China). The primer sequences used in this study are listed in Supplementary Table S2. The primers used in this study were synthesized by Shanghai Sangon Biotech Co. Ltd. or Eurofins Japan (Tokyo, Japan).