Part:BBa_K1628202

Pgrac

Promoter Pgrac is a promoter of lactose operon regulated by repressor LacI. Pgrac together with promoter PlacI, repressor LacI (BBa_K1628201), promoter Pxyl (BBa_K1628002) and repressor XylR (BBa_K1628203) formed a metabolic toggle switch. We used this device to regulate the expression of odhAB genes in Bacillus amyloliquefaciens NK-1 (showed in Figure 1).

We transformed the plasmids pHT01-xylR and pCB-Pxyl into the NK-1 strain, to verify the activity of metabolic toggle switch (see it on our wiki). Fresh colonies of Bacillus amyloliquefaciens strains (NK-1 strain containing plasmids pHT01-xylR and pCB-Pxyl and the control NK-1 strain containing plasmids pHT01 and pCB-Pxyl) were first cultured overnight in test tubes containing 5 mL LB liquid and then inoculated into 100 mL fresh fermentation medium. We added 1mM IPTG into the medium after 12h of cultivation. The β-galactosidase activity were measured at 12h, 18h, 24h, 30h, 36h, 42h to test the effect of metabolic toggle switch on the expression of bgaB.

As shown in Figure 2, β-galactosidase enzyme activity dropped considerably after 30 hours of fermentation. The inhibited expression of bgsB in experiment group (NPP+IPTG) indicated that the metabolic toggle switch we constructed is functional in B. amyloliquefaciens NK-1 strain.

NPU-CHINA 2021:Through the information searched by our team members[1], according to figure 3(A) pNDH33 and the DNA sequence of the Pgrac promoter (in capital letters) including the upstream AT-rich UP element, the lac operator (lacO; in capital letters) and the ribo-some-binding sequence (underlined) (showed in Figure 3)

NPU-CHINA 2021:According to the literature[2], we found Pgrac100 not only tightly controls the background expression level in E.coli, but also allowed high protein production levels at low IPTG concentrations(showed in Table 1).

2024 Hangzhou-BioX Team characterized this part for its ability to induce quorum-quenching in Bacillus subtilis against Aeromonas hydrophila

We demonstrated that Pgrac can control the expression of endogenous AHL lactonases in B. subtilis WB600.

AHL lactonases degrade AHLs, a class of signaling molecules that induce quorum sensing. Quorum sensing triggers the expression of virulence factors in pathogens, including A. hydrophila, an aquatic bacterium that threatens aquaculture (Coquant et al., 2020; Hlordzi et al., 2020). High expression of AHL lactonases in the probiotic B. subtilis reduces the virulence of A. hydrophila through quorum quenching and saves aquatic animals.

The quorum quenching abilities of Pgrac-controlled AHL lactonases (AiiA, YtnP, AttM, AiiM, AhlD, AhlS) were evaluated through synthetic AHL degradation tests (Figure 1), natural AHL degradation tests (Figure 2), biofilm reduction tests (Figure 3), and extracellular protease reduction tests (Figure 4).

Figure 1. A-C. Synthetic AHL degradation test on LB plates. B. subtilis cultures were loaded in wells. Liquid LB was used as the negative control. Rings without the purple color indicated AHL degradation; D. AHL degradation levels of each strain were measured in the width of the colorless ring; E. AHL degradation levels of mixed sample groups. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 2. A-C. Natural AHL degradation test on LB plates involved loading wells with a mixture of A. hydrophila and B. subtilis. LB served as the negative control. A. hydrophila alone and pure C4-HSL served as positive controls. Purple rings indicated the presence of C4-HSL; D. The AHL degradation levels of each strain were measured in the percentage reduction in ring size compared to the wells with A. hydrophila alone. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 3. Crystal violet biofilm assay. A. hydrophila was cultured in B. subtilis culture supernatants for 48 hours to allow biofilm formation. A. 48-hour A. hydrophila biofilm stained with crystal violet; B. Homogenized dye, prepared for OD570 measurement; C. Biofilm formation in each group was quantified using OD570/OD600; D. Biofilm formation in mixed sample groups. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 4. A. The activity of the extracellular proteases of A. hydrophila cultured in partial B. subtilis supernatants; B. Extracellular protease activities in mixed sample groups. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

The results showed that introducing all genes except AttM (BsAiiA, BsYtnP, MtAiiM, AsAhlD, and SsAhlS) under the control Pgrac enhanced the C4-HSL degrading ability of B. subtilis WB600. Among all groups, B. subtilis WB600 expressing AiiA under the control of Pgrac is the most effective quorum quenching strain against A. hydrophila, with the most decrease in biofilm formation (85.0%) and extracellular protease activities (42.96%), making it a promising probiotic for aquaculture applications.

We also demonstrated that the expression of AHL lactonases under the control of Pgrac in B. subtilis WB600 did not directly inhibit the growth of A. hydrophila, as quorum quenching does not kill bacteria but only affects the expression of specific virulence factors.

Figure 5. The inhibition test of A. hydrophila was conducted with wells loaded with B. subtilis. LB served as the negative control. Chloramphenicol (Cm+) served as the positive control. Clear rings around the wells indicated inhibition of A. hydrophila.

Reference

[1]Phan TT, Nguyen HD, Schumann W. Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis. Protein Expr Purif. 2006 Apr;46(2):189-95. doi: 10.1016/j.pep.2005.07.005. Epub 2005 Aug 9. PMID: 16125412.

[2]Phan, Trang & Linh Thuoc, Tran & Schumann, Wolfgang & Nguyen, Hoang. (2015). Development of Pgrac100-based expression vectors allowing high protein production levels in Bacillus subtilis and relatively low basal expression in Escherichia coli. Microbial cell factories. 14. 72. 10.1186/s12934-015-0255-z.

Coquant, G., Grill, J. P., & Seksik, P. (2020). Impact of N-Acyl-Homoserine Lactones, Quorum Sensing Molecules, on Gut Immunity. Front Immunol, 11, 1827. https://doi.org/10.3389/fimmu.2020.01827

Hlordzi, V., Kuebutornye, F. K. A., Afriyie, G., Abarike, E. D., Lu, Y., Chi, S., & Anokyewaa, M. A. (2020). The use of Bacillus species in maintenance of water quality in aquaculture: A review. Aquaculture Reports, 18, 100503. https://doi.org/https://doi.org/10.1016/j.aqrep.2020.100503

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]