Team:NKU CHINA/Demonstrate
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Demonstrate
1. PgltAB can be down regulated by glutamate
PgltAB-GFP and P43-GFP were transformed into both B. amyloliquefaciens LL3 Δbam and B. amyloliquefaciens LL3 Δbam -icd strain (with stronger promoter before icd gene), which were designated as LL3-PgltAB-GFP and LL3-icd-PgltAB-GFP respectively. The two mutants were cultured in M9 culture medium for 24 hours. If needed the medium was supplemented with antibiotics or glutamate at the following concentrations: 5 µg/mL chloramphenicol, 0 g/L, 0.5 g/L, 1.0 g/L, 2.5 g/L or 5.0 g/L glutamate. During the fermentation, 1.5mL bacteria culture was taken out every 3 hours, of which 600µL was for GFP FI measurement (395nm\509nm) by microplate reader, and 900µL for OD600 measurement.
With the extracellular glutamate concentration increasing, the FI of GFP was decreasing, which means higher glutamate concentration can indeed repress the promoter PgltAB's effect. The FI first rose and then fell, which may due to the extra glutamate adding that can promote cell growth. (Fig. 1.)
Fig.1 FI of GFP in LL3-PgltAB-GFP and LL3–icd-PgltAB-GFP under different extracellular glutamate concentrations in plateau stage. a. The intracellular glutamate concentration under different extracellular glutamate concentrations in plateau stage. The value illustrates the relationship between glutamate concentration in medium and intracellular glutamate concentration. *Significantly different (P < 0.05) by Student's t-test. b. FI of GFP in LL3-PgltAB-GFP under different extracellular glutamate concentrations in plateau stage. **Very significantly different (P < 0.01) by Student's t-test. c. FI of GFP in LL3-icd-PgltAB-GFP under different extracellular glutamate concentrations in plateau stage. *** Very very significantly different (P < 0.005) by Student's t-test. The strains were cultured at 37 °C in M9 medium with 5 µg/mL chloromycetin for 24 hours under different extracellular glutamate concentration (0 g/L, 0.5 g/L, 1.0 g/L, 2.5 g/L, 5.0 g/L). Intracellular glutamate concentration, fluorescence intensity of GFP and the OD600 were measured. FI of GFP was normalized against OD600. Data indicate mean values ± standard deviations from three independent experiments performed in triplicates.
2. High intracellular glutamate concentration promoted tetA expression
To test the expression of tetA, we tagged it with the fluorescent reporter GFP-coding gene (BBa_K2705004), whose expression was detected by microplate assay (395nm\509nm). The intracellular glutamate concentration and bacteria concentration (OD600) were also examined, respectively. (See Figure 2.) It could be concluded that with the increasing glutamate in medium, intracellular glutamate concentration went high, and tetA of PopQC was upregulated to express. The results suggested that the system can help individuals with higher intracellular glutamate concentration express more TetA, so that be able to survive in the tetracycline condition.
Figure 2. The relationship of extra- and intra-cellular glutamate concentration and FI of GFP /OD600 in plateau stage. The strains were cultured in fermentation medium with 10 µg/mL tetracycline for 24 hours. Intracellular glutamate concentration, OD600 and FI of GFP were measured in plateau stage. Data indicate mean values ± standard deviations from three independent experiments performed in triplicates.a. The relationship between extra- and intracellular glutamate concentration. The value illustrates the relationship between glutamate concentration in medium and intracellular glutamate concentration. b. The relationship between intracellular glutamate concentration and FI of GFP /OD600 in Plateau stage. The value illustrates the effect of glutamate concentration on GFP fluorescence intensity, which is normalized against OD600. *Significantly different (P < 0.05) by Student's t-test.
3. Population Quality Control (PopQC) System improving the yield of poly-γ-glutamate (γ-PGA)
The original intention of our project is to improve the yield of target metabolite using the PopQC system. We took γ-PGA as an example and measured the γ-PGA yield in both the previous constructed B. amyloliquefaciens NK-Ipop, which contained the pHT01-mCherry-LacI-PgltAB-Pgrac-TetA-GFPint plasmid, and the wild type strain. It can be seen that γ-PGA yield of the NK-Ipop was significantly higher than that of the wild strain. (Fig. 3)
Fig. 3 The γ-PGA yield of B. amyloliquefaciens NK-Ipop and LL3 Δbam. B. amyloliquefaciens NK-Ipop was cultured in the fermentation medium with 10 μg/mL tetracycline for 24 hours. and the wild strain was cultured in the fermentation medium without antibiotics. Data indicate mean values ± standard deviations from three independent experiments performed for B. amyloliquefaciens NK-Ipop, 2 independent experiments performed for B. amyloliquefaciens LL3 Δbam.
4. Safety
Taking safety into consideration, we must ensure that none of our microbe can grow outside of lab in case of leaking out. We verified our Asp auxotroph in B. amyloliquefaciens LL3 which was constructed by a markerless gene replacement method in order to delete its original pathways of synthesizing Asp. We provided the same medium without Asp to both Asp auxotroph and wild type of B. amyloliquefaciens LL3, the result indicated that Asp auxotroph couldn’t grow in Asp-free medium while the wild type cangrew normally.
Fig. 4 Asp auxotroph mutant's growth condition in the medium with or without Asp. The Asp auxotroph mutant was cultured in M9 medium with or without asparagine, separately. 2 independent experiments were carried out.
1. PgltAB can be down regulated by glutamate
PgltAB-GFP and P43-GFP were transformed into both B. amyloliquefaciens LL3 Δbam and B. amyloliquefaciens LL3 Δbam -icd strain (with stronger promoter before icd gene), which were designated as LL3-PgltAB-GFP and LL3-icd-PgltAB-GFP respectively. The two mutants were cultured in M9 culture medium for 24 hours. If needed the medium was supplemented with antibiotics or glutamate at the following concentrations: 5 µg/mL chloramphenicol, 0 g/L, 0.5 g/L, 1.0 g/L, 2.5 g/L or 5.0 g/L glutamate. During the fermentation, 1.5mL bacteria culture was taken out every 3 hours, of which 600µL was for GFP FI measurement (395nm\509nm) by microplate reader, and 900µL for OD600 measurement.
With the extracellular glutamate concentration increasing, the FI of GFP was decreasing, which means higher glutamate concentration can indeed repress the promoter PgltAB's effect. The FI first rose and then fell, which may due to the extra glutamate adding that can promote cell growth. (Fig. 1.)
Fig.1 FI of GFP in LL3-PgltAB-GFP and LL3–icd-PgltAB-GFP under different extracellular glutamate concentrations in plateau stage. a. The intracellular glutamate concentration under different extracellular glutamate concentrations in plateau stage. The value illustrates the relationship between glutamate concentration in medium and intracellular glutamate concentration. *Significantly different (P < 0.05) by Student's t-test. b. FI of GFP in LL3-PgltAB-GFP under different extracellular glutamate concentrations in plateau stage. **Very significantly different (P < 0.01) by Student's t-test. c. FI of GFP in LL3-icd-PgltAB-GFP under different extracellular glutamate concentrations in plateau stage. *** Very very significantly different (P < 0.005) by Student's t-test. The strains were cultured at 37 °C in M9 medium with 5 µg/mL chloromycetin for 24 hours under different extracellular glutamate concentration (0 g/L, 0.5 g/L, 1.0 g/L, 2.5 g/L, 5.0 g/L). Intracellular glutamate concentration, fluorescence intensity of GFP and the OD600 were measured. FI of GFP was normalized against OD600. Data indicate mean values ± standard deviations from three independent experiments performed in triplicates.
2. High intracellular glutamate concentration promoted tetA expression
To test the expression of tetA, we tagged it with the fluorescent reporter GFP-coding gene (BBa_K2705004), whose expression was detected by microplate assay (395nm\509nm). The intracellular glutamate concentration and bacteria concentration (OD600) were also examined, respectively. (See Figure 2.) It could be concluded that with the increasing glutamate in medium, intracellular glutamate concentration went high, and tetA of PopQC was upregulated to express. The results suggested that the system can help individuals with higher intracellular glutamate concentration express more TetA, so that be able to survive in the tetracycline condition.
Figure 2. The relationship of extra- and intra-cellular glutamate concentration and FI of GFP /OD600 in plateau stage. The strains were cultured in fermentation medium with 10 µg/mL tetracycline for 24 hours. Intracellular glutamate concentration, OD600 and FI of GFP were measured in plateau stage. Data indicate mean values ± standard deviations from three independent experiments performed in triplicates.a. The relationship between extra- and intracellular glutamate concentration. The value illustrates the relationship between glutamate concentration in medium and intracellular glutamate concentration. b. The relationship between intracellular glutamate concentration and FI of GFP /OD600 in Plateau stage. The value illustrates the effect of glutamate concentration on GFP fluorescence intensity, which is normalized against OD600. *Significantly different (P < 0.05) by Student's t-test.
3. Population Quality Control (PopQC) System improving the yield of poly-γ-glutamate (γ-PGA)
The original intention of our project is to improve the yield of target metabolite using the PopQC system. We took γ-PGA as an example and measured the γ-PGA yield in both the previous constructed B. amyloliquefaciens NK-Ipop, which contained the pHT01-mCherry-LacI-PgltAB-Pgrac-TetA-GFPint plasmid, and the wild type strain. It can be seen that γ-PGA yield of the NK-Ipop was significantly higher than that of the wild strain. (Fig. 3)
Fig. 3 The γ-PGA yield of B. amyloliquefaciens NK-Ipop and LL3 Δbam. B. amyloliquefaciens NK-Ipop was cultured in the fermentation medium with 10 μg/mL tetracycline for 24 hours. and the wild strain was cultured in the fermentation medium without antibiotics. Data indicate mean values ± standard deviations from three independent experiments performed for B. amyloliquefaciens NK-Ipop, 2 independent experiments performed for B. amyloliquefaciens LL3 Δbam.
4. Safety
Taking safety into consideration, we must ensure that none of our microbe can grow outside of lab in case of leaking out. We verified our Asp auxotroph in B. amyloliquefaciens LL3 which was constructed by a markerless gene replacement method in order to delete its original pathways of synthesizing Asp. We provided the same medium without Asp to both Asp auxotroph and wild type of B. amyloliquefaciens LL3, the result indicated that Asp auxotroph couldn’t grow in Asp-free medium while the wild type cangrew normally.
Fig. 4 Asp auxotroph mutant's growth condition in the medium with or without Asp. The Asp auxotroph mutant was cultured in M9 medium with or without asparagine, separately. 2 independent experiments were carried out.