Kanamycin

The amino acid sequences encoding Atezolizumab (Drugbank accession number: DB11595) were codon optimized in silico using GeneArt gene synthesis software (Thermo Scientific, MA, USA) for the expression of anti-PD-L1 antibody in plant system. Variable regions of Atezolizumab light chain (LC) and heavy chain (HC) that were added individually to human IgG₁ kappa chain (Genbank accession number: AAA58989.1) and gamma chain (Genbank accession number: AAA02914.1) were flanked with signal peptide on the N-terminus and a SEKDEL (Ser-Glu-Lys-Asp-Glu-Leu) sequence on C-terminus. Anti-PD-L1 light chain (anti-PD-L1-LC) and anti-PD-L1 heavy chain (anti-PD-L1-HC) that are synthesized were used for cloning into the geminiviral vector pBYR2eK2Md (pBYR2e) by double digestion with XbaI and SacI restriction enzymes (BioLabs, MA, USA) separately (Fig 1). The constructed plant expression vectors were transformed into Escherichia coli DH10B by heat shock method. Selected colonies were confirmed by colony polymerase chain reaction (PCR) with the primers mentioned in Table 1 and further by sequencing. Confirmed clones were cultured in Luria Bertani (LB) media (HiMedia Laboratories, Mumbai, India) with 50 μg/mL kanamycin (AppliChem, Dermstadt, Germany) overnight at 37°C and the plasmids were isolated and transformed into Agrobacterium tumefaciens GV3101 strain by electroporation.

The bacterial strains and plasmids used in this study are listed in Table 1. All bacteria were cultured in lysogeny broth (LB; 10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl) or on LB agar (15 g/l agar) at 37°C except Serratia plymuthica, which was grown at 30°C. Media were supplemented with the following chemicals (Applichem, Darmstadt, Germany) when appropriate: 5 g/l glucose; 100 μg/ml ampicillin (Ap); 200 μg/ml carbenicillin (Cb); 30 μg/ml chloramphenicol (Cm); 5 μg/ml gentamicin (Gm); 10 μg/ml tetracycline (Tc); 50 μg/ml kanamycin (Km); and 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). Plasmids pTrc99A and pTrc99A-P_trc-budAB were introduced into the mixed-acid fermenters by electroporation. All oligonucleotides used in this work are listed in Table 2, and were purchased from IDT (Haasrode, Belgium).

The SARS-CoV-2 spike protein-coding plasmid was kindly provided by Professor Gary Kobinger (University of Laval, Québec City, QC, Canada).
Plasmid DNA was amplified using Subcloning Efficiency DH5α Competent Cells (Thermo Fisher Scientific, Waltham, MA, USA, 18265017) grown in LB medium containing 100 μg/mL ampicillin (AppliChem, Darmstadt, Germany, A0839) and 100 μg/mL kanamycin (AppliChem A1493). Plasmid DNA was then purified using an EndoFree Plasmid Mega Kit (Qiagen, Hilden, Germany, 12381).
The amplified plasmid was finally analyzed by EcoRI digestion followed by gel electrophoresis, and was also fully sequenced using the iGE3 Genomics Platform of the University of Geneva using Illumina HiSeq 4000 technology.

In all experiments, Salmonella Typhimurium SL1344 (S.Tm;SB300; SmR) or the indicated ssaV mutant version (S.Tm^ssaV; M2730; AmpR) were used. [44 (link),45 (link)]. WITS-tags were introduced into S.Tm by P22 phage transduction and subsequent selection on kanamycin. The presence of the correct WITS-tag was confirmed by PCR using tag-specific primers [14 (link),46 (link)]. For in vivo mouse infections, bacteria were grown in lysogeny broth (LB) containing the appropriate antibiotics (50 μg/ml streptomycin (AppliChem); 15 μg/ml chloramphenicol (AppliChem); 50 μg/ml kanamycin (AppliChem); or 100 μg/ml ampicillin (AppliChem)) at 37°C for 12h and sub-cultured in 1:20 LB without antibiotics for 4h. Cells were washed and re-suspended in cold PBS (BioConcept).

In all experiments, Salmonella Typhimurium SL1344 (SB300, Sm^R) or the indicated mutant versions were used (summarized in Table 1). Desired genetic constructs were transferred into the appropriate background strain using P22 HT105/1 int-201 phage transduction [79 (link)]. Antibiotic resistance cassettes were removed using the heat inducible FLP recombinase encoded on pCP20, if desired [80 (link)]. For in vivo mouse infections, bacteria were grown in lysogeny broth (LB) media containing the appropriate antibiotics (50 μg/ml streptomycin (AppliChem); 15 μg/ml chloramphenicol (AppliChem); 50 μg/ml kanamycin (AppliChem); 100 μg/ml ampicillin (AppliChem)) at 37°C for 12 h and subcultured in 1:20 LB without antibiotics for 4 h. Cells were washed and resuspended in cold PBS (BioConcept).

Luria-Bertani broth (LB) and LB agar were purchased from Roth (Karlsruhe, Germany). The 5×M9-minimal salt contained di-sodium hydrogen phosphate (33.9 g/L; Roth, Karlsruhe, Germany), potassium-dihydrogen phosphate (15 g/L; Merck, Darmstadt, Germany), sodium chloride (2.5 g/L; Merck, Darmstadt, Germany) and ammonium-chloride (5 g/L; Merck, Darmstadt, Germany). This M9-salt was diluted 1:5 and supplemented with magnesium sulfate (1 mM, Merck, Darmstadt, Germany), calcium chloride (0.1 mM; Merck, Darmstadt, Germany), glucose (0.4%, Merck, Darmstadt, Germany), thiamin-hydrochloride (10 µg/mL, Roth, Karlsruhe, Germany) and nicotinic acid (0.0025%, Roth, Karlsruhe, Germany). We bought McCoy and RPMI-1640 from Merck (Darmstadt, Germany) and FBS from Thermo Fisher Scientific (Waltham, MA, USA). Human urine was donated by healthy individuals. The urine was filtered (0.2 µm pore size) and used at the day of donation. Potassium chloride came from Merck (Darmstadt, Germany), potassium sulfate from Roth (Karlsruhe, Germany). Phorbol 12-myristate 13-acetat was bought from PromoCell (Heidelberg, Germany). We purchased E. coli LPS K235 from Merck (Darmstadt, Germany), kanamycin, ampicillin and gentamycin from AppliChem (Darmstadt, Germany) and chloramphenicol from Merck (Darmstadt, Germany).

Bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli strain DH5α was routinely used for plasmid propagation and cloning experiments, and cultivated on Luria-Bertani (LB) medium (Becton–Dickinson, Sparks, MD, USA) supplemented with 100 mg/L ampicillin (AppliChem, Darmstadt, Germany) at 37 °C. S. aureus strains were grown with aeration in trypticase soy broth (TSB) (Difco Laboratories, Detroit, MI, USA) in a rotating incubator (at 180 rpm) at 37 °C. If required, tetracycline and kanamycin (AppliChem) were added at a final concentration of 10 mg/L. Oxacillin (Sigma-Aldrich, Buchs, Switzerland) was used at 4 mg/L, which corresponded to the MICs for strain N315.Table 1

Bacterial strains and plasmids

Strain or plasmid	Relevant characteristics	References
Strains
E. coli DH5α	Host for DNA cloning	Laboratory collection
S. aureus
RN4220	Restriction-deficient derivative of S. aureus RN450	[11 (link)]
N315	MRSA carrying type II SCCmec	[12 (link)]
N315EX	Isogenic MSSA derivative of N315	This study
Plasmids
pUC28	ColE1 replicon, high copy number vector for cloning, ampicillin resistance	[13 (link)]
pSR3-1	Thermosensitive-replicon plasmid carrying the ccrAB genes of strain N315 (used for SCCmec excision in N315), tetracycline resistance	[7 (link)]
qPlasmid	Plasmid used for qPCR analysis	This study