# Purification and Characterization of Authentic 30S Ribosomal Precursors Induced by Heat Shock
## 4 Materials and Methods
### Tagging of uS2 and bS20  
The primers rpsTREPF-rpsTREPR (for _rpsT_) and rpsBHISF-rpsFR (for _rpsB_) were used to amplify the KanR gene from pKD4 [24]. The PCR products were then recombined into the \begin{table}
\begin{tabular}{l l l} \hline \hline
**Strains** & \\ \hline MG1655 & F\({}^{-}\)\(\lambda^{-}\)_rph_ 1 & CGSC 6300 [48] \\ \hline E5990 & MG1655 _rpsB_:his-KanR & This study \\ E6001 & MG1655 _rpsB_:his & This study \\ \hline E5998 & MG1655 _rpsT_:strep-kanR & This study \\ \hline E6004 & MG1655 _rpsT_:strep & This study \\ \hline E6006 & MG1655 _rpsB_ his _rpsT_ strep & This study \\ \hline
**Plasmids** & \\ \hline pKD46 & \begin{tabular}{l} repA101(Ts) _araB_p-_gam-_net-exo_ \\ ori101 AmpR _araC_ \\ \end{tabular} & [24] \\ \hline pKD4 & \
\begin{tabular}{l} oriR6 Kgamma AmpR _rguB_ \\ KanR flanked by FRT \\ \end{tabular} & [24] \\ \hline pCP20 & Rep Ts Cl857 AmpR CmR FLP & [24] \\ \hline pBAD24 &
\begin{tabular}{l} oriCoEl 1 araC _araB_p \\ \end{tabular} & [49] \\ \hline pE7867 & pBAD24 rpsT:strep & This study \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Strains and plasmids. \begin{table}
\begin{tabular}{l l l} \hline \hline
**Type** & **Sequence** & **Experiment** \\ \hline \multirow{3}{*}{rpsTREPF} & GCACGTCATAAGGCTAACCTGACTGCACAGATC & \multirow{3}{*}{rpsT strep} \\  & AACAACTGGCTTGAGCCACCCGACTTGCAA & \\  & AAGTAAGGTTAGGCCTGGACTGTTC & & \\ \hline \multirow{3}{*}{rpsTREPR} & AACCGCCTTGCCCGGCGTTTTTCACAAAGCTTCA & \multirow{3}{*}{rpsT strep} \\  & GCAAAATTGGCCGAAATTGGCAATTGGCC & \\ \hline \multirow{3}{*}{rpsBHISF} & CAGGATCTGGTCCACGGGGAAAAAGCTTCG & \multirow{3}{*}{rpsB his} \\  & TAGAACGTAGACCACCACCACCATA & & \\ \cline{1-1}  & GTGTAGGCTGGAGCTGTTC & & \\ \hline \multirow{3}{*}{rpsFR} & GCCTTTCTGCAAACTGAACTATTGGGGAGT & \multirow{3}{*}{rpsB his} \\  & TATAAGCCTTAAATGGGAATTAGCCATGGTCC & \\ \hline pkd4F & AAGCCATCCAGTTACTTTGC & & KanR insertion \\ \hline pkd4D & CGCATGCCTTCTATCC & & verification \\ \hline rpsBD & GCTTCTCAGGATTGGCTTC & & rpsT mutation \\ \hline rpsBF & CTCGGATGATTGGCC & & Verification \\ \hline rpsTD & GTAAGCCAAACGCCAAGCCG & & rpsT mutation \\ \hline rpsTF & ACAGACCACTGGAGCCAC & & Verification \\ \hline \multirow{3}{*}{rpsTA} & TTTTCCATGCTAATATCAAATCAGCTAAGA & \multirow{3}{*}{
\begin{tabular}{l} cloning of rpsT \\ ORF \\ \end{tabular} } \\  & GCCC & & \\ \hline
21S-115 & 5\({}^{\prime}\) Biotin-TEG CATTTTTCGGTGTGCGACG & \multirow{3}{*}{detection of 17S} \\ \cline{1-1}  & 5\({}^{\prime}\) Biotin-TEG CAAAGAACGCCTTTTAAG & & \\ \hline
16S-F27C & 5\({}^{\prime}\) Biotin-TEG CTGGACCATGAAACTCT & & \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Oligonucleotides used. _E. coli_ MG1655 chromosomes using their protocol. MG1655 carrying the pKD46 plasmid was grown in LB medium with ampicillin and L-arabinose at 30 \({}^{\circ}\)C to an approximate OD\({}_{600}\) 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 \(\mu\)F, 200 \(\Omega\)) with 0.25-cm chambers. Transformants were selected on LB-kanamycin plates at 42 \({}^{\circ}\)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 \({}^{\circ}\)C, incubated at 42 \({}^{\circ}\)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.