coli’ pathway-based proteolytic system in E. coli was performed using homologous recombination technology. Using the strain constructed in ‘Replacement of the tnaA gene with the trpR gene’, the DNA fragment consisting of Ptrp, learn more the kan gene, and ORF of the ubi4 gene was inserted in-frame upstream of the target gene (dnaB, fab, or pyrG) originally present in the chromosome
by homologous recombination (Product No. 33, 34, 60 in Table S2 and Fig. 1a). For the construction of the GlmU, DnaX, DnaG, IspA, Era, PyrH, or Der, we used a construction strategy that can be applied in cases where direct recombination of the essential target gene (the method described in ‘Construction of the bacterial strain targeting DnaB,FabB, or PyrG’) is unsuccessful or if the target gene has an operon structure. Using the strain constructed in ‘Replacement of the tnaA gene with the trpR gene’, the DNA fragment of ORF of the ubi4 gene fused
in-frame to ORF of the target gene (glmU, dnaX, dnaG, der, pyrH, era, or ispA) was inserted downstream of the tryptophan promoter present in the chromosome by homologous recombination, and then each original gene in the chromosome was deleted by homologous recombination with DNA fragment encoding the kan gene (Fig. 1b). Colony formation Ganetespib manufacturer assay was performed using the strains
constructed in ‘Construction of tryptophan promoter-dependent expression system in E. coli’ (Table 2). No colony was detected in the LB agar plate containing Trp (1 mg mL−1) alone or containing Trp (1 mg mL−1) plus IPTG (10 mM), under the condition that colony (-)-p-Bromotetramisole Oxalate formation (about 1000 CFU per plate) was observed in the LB agar plate containing IAA (25 μg mL−1). This result suggested that the Trp-mediated inhibition of the target gene expression was sufficient for evaluation of the colony-forming capacity as a phenotype. When the strain targeting DnaB was examined, colonies were detected in the LB agar without any supplement, suggesting that these strains do not require DnaB at the higher expression level induced by IAA for colony formation. By contrast, the number of colonies was not altered by inducing the proteolytic system alone in the strains targeting DnaB, although the size of colonies was very small (data not shown). In strains in which the tnaA gene was not replaced with the trpR gene, the inhibition of colony formation was not observed in the LB agar plates containing Trp, suggesting that the tryptophanase activity of endogenous TnaA interferes with the Trp-mediated inhibition of Ptrp in this system (data not shown).