Existing part: BBa_K118011 New part: BBa_K4115004

We construct intercellular genetic circuit feedback to regulate the nutrient flux in our ternary microbial symbiosis system. In the genetic circuit, starvation promoters in E. coli are sensors for nutrient signals. To ensure a good signal-to-noise ratio for signal input, we made efforts on improving the properties of the starvation promoters.

We evaluate the quality of promoters from two aspects. First, promoter activity is an important evaluation index. Empirically, we believe that promoter activity not lower than the J23101 (a constitutive promoter with moderately strong activity) is necessary for production and genetic circuits. Second, fold-change is another important perspective for promoters, especially for those being used in complicated genetic circuits. Fold-change can be defined as the ratio of the activity in the activated state to the non-activated state. For our starvation promoters, the practical definition is the ratio of promoter activity under low glucose concentration to that under high glucose concentration.

We first selected three starvation promoters and test their response to low glucose concentrations. We construct reporter genes as the construct in Figure 1, and quantified the promoter activity with fluorescence intensity. Among the three promoters, PcstA had the largest fold-change and was comparable to the activity of J23101. However, compared with the widely used inducible promoters like pLac or pBAD, its activity and fold-change are not good enough. So we did several improvement cycles based on the 'Design-Build-Test-Learn' cycle.

Figure 1. sfGFP with LVA is used to indicate the activities of PcstA variants.

PcstA (BBa_K118011) is a well-characterized starvation promoter in iGEM Registry. PcstA uses RpoD as its sigma factor. cAMP receptor protein (CRP) is an activator of PcstA. When undergoes carbon source starvation conditions, the intracellular cAMP concentration will increase and further activate CRP. Then CRP binds to a CRP-binding sequence located upstream of the transcription start site and activates of transcription initiation of PcstA (Figure 2).

Figure 2. CRP-dependent activation of PcstA under starvation conditions.

Furthermore, PcstA is down-regulated by the factor for inversion stimulation (Fis) under nutrient-rich conditions (Figure 3). Under starvation conditions, the Fis abundance in cells will decrease, and the inhibition will be removed.

Figure 3. Fis-dependent inhibition of PcstA under nutrient-rich conditions.

To further optimize the activity and fold-change of PcstA, we made several mutations in its regulation-related sequence. We first mutate the CRP-binding sequence to improve the affinity of the promoter for CRP, so that CRP can activate the promoter more efficiently. The mutants constructed in the first cycle are shown in Figure 4. In principle, if the CRP-binding sequence of PcstA has higher similarity to the consensus sequence, PcstA will have a higher affinity to CRP. The consensus sequence of the CRP-binding site is AAATGTGA-N6-TCTCATTT. The nucleotides with underline are the core of the CRP-binding sequence. G and C with underlines provide most of the binding energy^[1]. Based on these rules, we constructed PcstA-Con and PcstA-TCACA to test what will happen if PcstA has a higher affinity to CRP.

Figure 4. The CRP-binding sequences of PcstA variants in the first cycle.

The reporter gene in Figure 1 is used to test the properties of these PcstA variants. Data is summarized in Figure 5. The promoter activity was significantly improved by the mutations. PcstA-Con has a 20 times higher activity compared with the original PcstA. So it's hard to indicate the data in a column figure. However, it seems that their affinities to CRP are too high. Even under a high glucose concentration of 4 g/L, they are still activated and show no response to starvation. In this cycle, we learned that high similarity with the consensus sequence is not always a good thing. There might be a suitable range for the affinities of regulators.

Figure 5. Summarized data of the relative activities and fold-changes of PcstA variants. The relative activities are normalized with FI/OD₆₀₀ of J23101 at 4g/L glucose.

Then we decide to mutate another site that has relatively little effect on affinity.

Figure 6. The CRP-binding sequences of PcstA variants in the second cycle.

The reporter gene constructs are cloned into pUC high-copy number backbone. A significant promoter activity increase was observed on PcstA_Mutant1 and the starvation response function remained (Figure 7A). However, some unwanted phenomena also appeared. First, the leakage expression of PcstA_Mutant1 under high glucose concentration increases, which results in the decrease of fold-change. Second, the high copy of PcstA_Mutant1 in cells has some side effects on growth (Figure 7B). Actually, the growth rate decrease was more obvious in PcstA-Con and PcstA-TCACA transformed strains. So we hypothesize that the high copy of PcstA_Mutant1 causes these side effects. If PcstA_Mutant1 keeps a high copy in cells, it may compete the CRP with the endogenous CRP-dependent promoter and influence the global metabolism.

Figure 7. (A) FI/OD₆₀₀ of PcstA and PcstA_Mutant1 (B) Growth curve of Empty and PcstA_Mutant1 strain in M9 minimal medium containing 4.0 g/L glucose as the sole carbon source.

At the end of the last cycle, we got a function variant, which has higher activity and remains the starvation response. To eliminate the undesirable properties of PcstA_Mutant1, we shifted all constructions to the low-copy number pET backbone and repeat the experiment (Figure 8). The bacteria growth is back to normal in the PcstA_Mutant1 group. PcstA_Mutant1 shows significantly higher activity as well as larger fold-change with low-copy number backbone. The data of relative promoter activities and fold changes of PcstA variants in this cycle are summarized in Figure 9 .

Figure 8. FI/OD (600) of PcstA and PcstA_Mutant1. All the constructs are cloned into pET low-copy number backbone.

In conclusion, by mutating the regulation-related sequence and replacing the plasmid backbone, we finally get Pcst_Mutant1 (on pET backbone) as the best construct for the nutrient response.

Figure 9. Summarized data of PcstA variants. High [Glucose]=2 g/L, Low [Glucose]=0.25 g/L. Relative promoter activities are normalized with FI/OD (600) of J23101 at 2 g/L glucose

[1] Soberon-Chavez, G., Alcaraz, L.D., Morales, E., Ponce-Soto, G.Y., and Servin-Gonzalez, L. (2017). The Transcriptional Regulators of the CRP Family Regulate Different Essential Bacterial Functions and Can Be Inherited Vertically and Horizontally. Front Microbiol 8. ARTN 95910.3389/fmicb.2017.00959.