Genetic Manipulation of Ribosomal Proteins

The primers rpsTREPF-rpsTREPR (for rpsT) and rpsBHISF-rpsFR (for rpsB) were used to amplify the KanR gene from pKD4 [24 (link)]. The PCR products were then recombined into the E. coli MG1655 chromosomes using their protocol. MG1655 carrying the pKD46 plasmid was grown in LB medium with ampicillin and L-arabinose at 30 °C to an approximate OD₆₀₀ of 0.6, then made electrocompetent [47 ]. PCR products were gel-purified, digested with DpnI, re-purified, and then suspended in 10 mM Tris (pH 8.0). Electroporation was performed according to the manufacturer’s instructions in a BIO-RAD Gene Pulser II (2.5 kV, 25 µF, 200 Ω) with 0.25-cm chambers. Transformants were selected on LB-kanamycin plates at 42 °C. To check for possible loss of the pKD46 plasmid, the colonies were tested for ampicillin sensitivity. The chromosomal structure of mutants was confirmed by PCR (Supplementary Figure S1). KanR colonies were then transformed with pCP20 plasmid. The resulting AmpR transformants were selected at 30 °C, incubated at 42 °C, and tested for antibiotic resistance. A PCR was also used to confirm the mutations of rpsB and rpsT (Supplementary Figure S1). The rpsB:his and rpsT:strep strains were named ‘E6001’ and ‘E6004’, respectively. The rpsB-KanR mutation was transduced into E6004. Transductants were selected as KanR colonies, and then pCP20 was introduced by transformation. The KanR-AmpR colonies were isolated and their chromosomal structures were confirmed by PCR. Finally, KanR was deleted after induction of Flp recombinase, resulting in an ‘E6006’ strain containing both rpsT:his and rpsB:strep.