Design

In our work, promoter P_gltAB (BBa_K2705000), promoter P_grac (BBa_K2705002), lacI (BBa_K2705001) gene and tetA (BBa_K2705003) gene were composed to build up the PopQC system. (See Figure 1.) The function of P_gltAB is documented in BBa_K2705000.

Fig. 1 Design principle of PopQC for high yield of γ-PGA. PopQC system selects non-genetic high-performing cells, which finally leads to high producers outcompete the low producers. In specific, we use the promoters P_gltAB and P_grac, lacI gene and tetA gene. The tetracycline efflux protein TetA, which responds to intracellular glutamate concentration via the promoters P_gltAB and P_grac, which respond to the LysR family regulator GltC and the lactose repressor LacI, separately. With tetracycline, high producers will dominant the population.

In Bacillus amyloliquefaciens LL3 exists the glt operon, which is responsible for intracellular glutamate synthesis (See BBa_K2705000 for more details about P_gltAB). TetA is a tetracycline resistance protein [TetA(C) inner-membrane-associated protein] (See BBa_K2705007 and BBa_K2705003 for more details about TetA).

With a specific extracellular tetracycline concentration, when intracellular glutamate-precursor of γ-PGA-concentration of the individual is low, GltC level will go up, which activates the P_gltAB to express lacI. LacI furthermore represses P_grac and as a result, represses tetA expression. On the contrary, for high-producers, the concentration of intracellular GltC will go down, which represses the P_gltAB to express lacI, and the tetA expression is not affected. Therefore, high-producers will synthesize enough amount of tetracycline efflux pumps to maintain alive while low-producers won’t be able to survive. Consequently, the average intracellular glutamate concentration among the population is enhanced, which will finally lead to γ-PGA yield enhancement in LL3.

Proof of Function

Vector pHT01-P_gltAB-LacI-P_grac-TetA was converted into LL3, and correct transformants were fermented in M9 culture medium with different extracellular glutamate concentrations (0, 2.5 and 5g/L). From the 6th hour, we tested bacteria with several assays every 3 hours. LL3 Δbam strain was chosen as control system.

Expression level of tetA by microplate assay

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.

Measurement of γ-PGA yield

After 24 hours fermentation, the γ-PGA yields of both strains without extra glutamate were tested. With PopQC system, NK-Ipop strain produced more γ-PGA, which approved the system function. See Figure 4.

Figure 4. γ-PGA yields of B. amyloliquefaciens LL3 Δbam with and without PopQC system. The strains were cultured in fermentation medium with 10 µg/mL tetracycline for 24 hours. Dry weight of γ-PGA and the OD₆₀₀ were measured. Data indicate mean values ± standard deviations from three independent experiments performed for PopQC, 2 independent experiments performed for LL3 Δbam.

Design

This part includes the promoter P_gltAB and green fluorescent protein, the expression of GFP is repressed by high level cellular glutamate because P_gltAB is upregulated by GltC, which is repressed by high intracellular concentration of glutamate. A restriction enzyme KpnI cutting site is added between P_gltAB and GFP.

The part was applied to test the repression function of GltC to P_gltAB, for which we fermented under different glutamate concentration and tested the fluorescence level changes in LL3.

Proof of Function

Detection of gltC transcription level in LL3-P_gltAB-GFP under different glutamate concentrations

LL3-P_gltAB-GFP was cultured in M9 medium with different extracellular glutamate concentrations. From the 6th hour, we extracted the total RNA of LL3-P_gltAB-GFP every 3 hours and tested the transcription of gltC together with the respective intracellular glutamate concentrations. Transcription level of gltC in plateau phase is shown in Fig. 1. It could be indicated that the transcription of gltC was repressed with the increasing intracellular glutamate concentration. Primers used in the assay are listed in Table 1.

Fig. 1. The intracellular glutamate concentration and the relative expression level of gltC in LL3 with P_gltAB-GFP in plateau stage. a. The intracellular glutamate concentration of LL3 with P_gltAB-GFP in plateau stage. *Significantly different (P < 0.05) by Student's t-test. b. The relative expression level of gltC in plateau stage. The value illustrates the effect of glutamate concentration on the transcription of gltC. ***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 under different extracellular glutamate concentration (0 g/L, 2.5 g/L, 5.0 g/L) for 24 hours. Data indicate mean values ± standard deviations from three independent experiments performed in triplicates.

GFP fluorescent intensity (FI) reports the P_gltAB function

P_gltAB-GFP and P₄₃-GFP were converted into both B. amyloliquefaciens LL3 Δbam and B. amyloliquefaciens LL3 Δbam -icd strain (with stronger promoter before icd gene), which were designated as LL3-P_gltAB-GFP and LL3-icd-P_gltAB-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 P_gltAB's effect. The FI first rose and then fell, which may due to the extra glutamate adding that can promote cell growth. (Fig. 2 and Fig. 3.)

Fig.2 Principle for detecting the P_gltAB function. Under high glutamate concentration, GltC level goes down, reducing the level of GFP.

Fig.3 FI of GFP in LL3-P_gltAB-GFP and LL3–icd-P_gltAB-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-P_gltAB-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-P_gltAB-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.

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