In order to verify the whole regulation pathway step by step, we divided it into several parts, including the following concepts:

1. Intracellular glutamate can inhibit the gltC expression;
2. GltC can activate the promoter P_gltAB;
3. LacI can inhibit the promoter P_grac;
4. High glutamate concentration can improve tetA expression.

At last we successively verified the first and the fourth concepts, based on which we achieved the final goal–– control population quality through non-genetic cell-to-cell variation.

According to previous research, B. amyloliquefaciens LL3 is a glutamic acid- independent poly-γ-glutamate (γ-PGA)-producing strain which was isolated from traditional fermented food. Its gltAB gene encodes the glutamate synthase. The increasing intracellular glutamate concentration has a negative effect on the expression of gltAB via modulating GltC activity.

As a result, the transcription of gltC should be inhibited while intercellular glutamate concentration increasing. We verified this concept by transformed our PopQC system pHT01-mCherry-LacI-P_gltAB-P_grac-TetA-GFP^int into B. amyloliquefaciens LL3. Then we measured its intracellular glutamate concentration and transcriptional level of gltC under different concentration of glutamate in fermentation medium. The results proved our assumption to be correct.

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.

On the basis of the above experiment, we intended to choose GFP as a signal for P_gltAB activity, so that we can easily measure the florescence intensity by plate reader, combining with the transcriptional level of gltC measured at the same time. Due to time constraints, we didn't proof this concept.

As we all know, LacI plays an important role in lac operon model, it can sufficiently inhibit the activity of its downstream promoters. For Bacillus, the promoter P_grac consists of the groE promoter; the lacO operator and the gsiB SD sequence, among which the
the lacO operator can be inhibited by the LacI protein. Due to time constraints, we didn't proof this concept.

High glutamate concentration can improve 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 (395 nm\509 nm). 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.