# Constitutive Expression of a Cytotoxic Anticancer Protein in Tumor-Colonizing Bacteria
## 2 Materials and Methods  
### Bacterial Strains, Plasmids, and Culture Conditions  
Bacterial strains and plasmids are listed in Table 1. _E. coli_ K-12 MG1655 and _S. enterica_ serovar Gallinarum clinical isolate (SG4021) were used as wild-type strains and cultured in Luria Bertani broth (LB, Difco Laboratories, Franklin Lakes, NJ, USA). The \(\Delta\)ppGpp _S._ Gallinarum (SG4023) was constructed from SG4021 using the \(\lambda\) red system to disrupt the relA and spoT genes as described previously [26]. The \(\Delta\)ppGpp _S._ Gallinarum (SG4030) was constructed by p22 phage transduction as described previously [27] using 10% N-acetyl-D-glucosamine in LB medium. To monitor rrnB P1 promoter activity, the prmBP1-gfpOVA plasmid was constructed by Gibson assembly as follows: first, the rrnB P1 promoter containing Fis-binding sites in the upstream activation region (\(-\)154\(+\)3) was amplified from the _E. coli_ K-12 MG1655 chromosome [28] and cloned into a reporter gene (_gfpOVA_) by replacing the katG promoter sequence in the pkatG:gfpOVA plasmid [29]. The rrnB P1 promoter replaced the _araBAD_ promoter sequence in the pSEC-TGF\(\alpha\)-PE38 plasmid [25] with the specific primer set listed in Table 2, generating the prmBP1-psp-TP plasmid. The balancedlethal system based on the glmS gene was introduced into prrmBP1-psp-TP in the clal site to maintain the plasmid in vivo [23; 27]. Plasmids were confirmed via DNA sequencing (Macrogen, Seoul, Republic of Korea). Each plasmid was introduced into _E. coli_ by heat shock or _Salmonella_ by electroporation. Bacteria strains carrying the plasmid were grown in LB medium with 1% NaCl at 37 \({}^{\circ}\)C with vigorous shaking. When necessary, ampicillin (Sigma-Aldrich, Darmstadt, Germany) was added at a concentration of 100 \(\upmu\)g/mL. To identify amino acids required for growth, wild-type \(S\). Gallinarum and \(\Delta\)ppGpp \(S\). Gallinarum were cultured on M9 Minimal Medium plates (Welgene Precision SolutionTM, Gyeongsan, Republic of Korea) supplemented with glucose (0.2 g/mL), thiamine (5 mg/mL), magnesium sulfate (1 M), calcium chloride (1 M), and a mixture of 19 amino acids (100 \(\upmu\)g/mL, each) with 1 omitted from 20 essential amino acids.  
\begin{table}
\begin{tabular}{l l l} \hline \hline \multicolumn{1}{c}{**Status**} & \multicolumn{1}{c}{**Description**} & \multicolumn{1}{c}{**References**} \\ \hline _Escherichia coli_ & & \\ \hline MG1655 & Wild type (with defects in ilvG, rfb50, and rph-1) & ATCC \\  & & [30; 31] \\ \hline EMP4002 & MG1655, prrmBP1-sfpOVA, \(Amp^{\prime}\) & This work \\ \hline _Salmonella enterica_ serovar Gallinarum & & \\ \hline SG4021 & Wild-type isolate, clinical & \\ \hline SG4023 & SG4021, \(ArelA,AspT\) & [26] \\ \hline SG4030 & SG4023, \(ArelA,AspT,Aglms:\(Karn^{\prime}\) & This work \\ \hline SMP4001 & SG4023, prrmBP1-sfpOVA, \(Amp^{\prime}\) & This work \\ \hline SMP4003 & SG4030, prrmBP1-psp-TP, \(glmS\)+, \(Amp^{\prime}\) & This work \\ \hline SMP4004 & SG4030, pSEC-TGFa-PE38, \(glmS\)+, \(Amp^{\prime}\) & This work \\ \hline Plasmids & & \\ \hline prrmBP1-sfpOVA & \(\mathit{gfPOVA}\) under control of PrrmBP1 in pBR322 & This work \\ \hline prrmBP1-psp-TP & psp-TP under control of PrrmBP1 in pBAD24 & This work \\ \hline pSEC-TGFa-PE38 & psp-TP under control of PrrmBAD in pBAD24 & [25] \\ \hline \hline \end{tabular}
\end{table}
Table 1: Bacterial strains and plasmids used in this study.  
\begin{table}
\begin{tabular}{l l} \hline \hline \multicolumn{1}{c}{**Construction**} & \multicolumn{1}{c}{**Name and Direction *} & \multicolumn{1}{c}{**Sequence **} \\ \hline \multirow{6}{*}{prrmBP1-sfpOVA} & GFP-vector-FW & 5\({}^{\prime}\)-CGGAAAACTCCCTATAATGCGCGCACC \\  & ACTTCTAGATITAAGAAGGAGATATATICA-3\({}^{\prime}\) \\  & GFP-vector-RV & 5\({}^{\prime}\)-AAACCGTGAAAGGAGGACATAATTATTGTACGC \\  & GCF-vector-RV & 5\({}^{\prime}\)-AGCCGCAT