Q5 high fidelity 2 master mix

Unless otherwise
stated, all PCR
reactions were performed using a Q5 High-Fidelity 2× Master Mix
(New England Biolabs, M0492S) according to the manufacturer’s
instructions. For a single PCR reaction using four primers simultaneously,
the reaction mix was composed of 0.5 μM of each adapter primer,
0.025 μM each core primer (20× less concentrated than adapter
primers), 40–60 ng of DNA template, and 1× Q5 High-Fidelity
Master Mix. PCR conditions are described in Table S10. Cell-free backbone plasmid was synthesized by IDT (Integrated
DNA Technologies) using kanamycin as a resistance marker. New plasmids
were constructed using conventional PCR products or double-stranded
DNA fragments (Genewiz, U.K.) along with the destination pFGC-T7-RJBB
plasmid (vector backbone with the T7 promoter-RiboJ- BsaI-LacZα-BsaI-T7 terminator configuration)
in a single Golden Gate cloning reaction. All of the molecular cloning
steps and plasmid propagation were performed in E.
coli Top10 (Invitrogen, C404010). DNA plasmid for
cell-free reactions was obtained by midi-prepping (Sigma-Aldrich,
NA0200-1KT) an overnight culture of 50 mL LB with the appropriate
strain and antibiotic according to the fabricant’s instructions.
Plasmids and primers are listed in Tables S9 and S11 respectively. Coding sequences cloned into the pFGC-T7-RJBB
plasmid are described in Table S12. Plasmids
are available at Addgene (173224-27).

The bacterial strains and plasmids used in this study are listed in Tables S1 and S2, respectively. PCR was performed by KOD Hot Start DNA polymerase (National England BioLabs/NEB) or Q5 High-Fidelity 2× Master Mix (NEB) for cloning purposes according to the manufacturer’s instructions. Plasmid DNA and PCR fragments were purified using the plasmid miniprep kit (NEB) and DNA gel extraction kit (NEB). E. coli K12 chromosomal DNA was used as the template. E. coli DH5α stain was used for cloning, and E. coli BL21 (DE3) was used for recombinant protein purification. E. coli MG1655 strain and KO mutants were used for growth curve measurement, PG composition analysis, immunoblotting, and MIC study.
Unless otherwise specified, bacteria were cultured in LB [1% tryptone, 0.5% yeast extract, 0.5% NaCl] or LB-salt–free [1% tryptone, 0.5% yeast extract] medium at 37 °C. Agar 1.5% (w/v) was used in solid plates. Antibiotics were used at the following concentrations (per mL): 30 (chloramphenicol; Cm30), 50 (kanamycin; Kan50), or 100 (ampicillin; Amp100) where appropriate.

The isolation of genomic DNA and plasmid preparation using E. coli or S. aureus were performed with a bacterial genomic DNA kit and PurePlasmid Mini Kit, respectively (CwBIO, Jiangsu, China). Oligonucleotide primers were synthesized by Sangon Biotech (Shanghai, China). All primers used in this study are listed in Supplementary Table 2. For analytical purposes, PCRs were performed using OneTaq 2 × Master Mix (NEB, Ipswich, England). PCRs for plasmid construction were performed using Q5 High-Fidelity 2 × Master Mix from NEB according to the manufacturers’ instructions. The PCR products were purified using the DNA Clean-up Kit (CwBIO, Jiangsu, China). For cloning or plasmid construction, the plasmid was linearized by the related restriction enzymes from NEB. Cloning was performed using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China), which is based on homologous recombination.

The annealed DNA samples were modified by enzymes in different reaction systems. (i) Q5 DNA polymerase: 9 μl DNA sample in buffer A was mixed with 9 μl Q5 High-Fidelity 2× Master Mix (or 9 μl 2× Q5 Reaction Buffer as in a negative control) (NEB) for an incubation at 37°C for 5 h. (ii) T4 DNA ligase: 1 μl of T4 ligase was mixed with 20 μl annealed DNA sample in buffer B for an incubation at 16°C for 17 h. Effector staples were subjected to phosphorylation by T4 PNK (NEB) before ligation treatment.