Module 1: AAF Sensing Circuits

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The AAF sensing module mainly comprises two genetic circuits: an AND-gate sensing circuit and an Amplification & Feedback circuit. The AND-gate circuit enables the bacterial strain to sense changes in the intestinal environment, thereby flexibly regulating GLP-1 synthesis in a spatiotemporal manner. Then, the amplification and feedback circuit amplifies the upstream AND-gate signal to increase the synthesis level of GLP-1, thereby overcoming enzymatic degradation and transmembrane loss in the intestine. Additionally, it regulates GLP-1 levels based on feedbacks related to feeding and fasting states, alleviating the metabolic burden on engineered Escherichia coli (E. coli).

Due to limited timeframe and metabolic pressure of strains, we only performed separate validation for the two parts, optimized their expression conditions, gradually characterized the functions of the elements and logic circuits, and used the output signal intensity of the green fluorescent reporter protein (GFP) to relatively quantify the synthesis level of GLP-1.

Circuit 1: AND-gate Sensing

1.Characterization and optimization of two AND-gate input-inducible operons separately

We first verified the induced expression levels of the protocatechuic acid (PCA)-responsive and bile salt (BS)-responsive operons. After obtaining the pSB3K3 backbone plasmid with kanamycin resistance (Kan^R) and gene GFP(S65T) from Professor Baojun Wang's lab, we commissioned a company to synthesize the PCA-responsive operon and BS-responsive operon sequence. These two operons were then ligated with GFP(S65T) and the pSB3K3 backbone. Their respective fluorescent expression levels were characterized, and the combination of promoters and RBS was optimized to achieve the best induction effect.

(1)Construction of expression plasmids for the PCA-responsive operon

We obtained the GFP fragment with 18bp homology arms and the pSB3K3-J23117-PcaV-P_LV fragment from the plasmids we got via primer design and PCR amplification, with preliminary fragment verification conducted using agarose gel electrophoresis. The complete characterization plasmid was then constructed by Gibson Assembly, followed by transformation into E. coli TOP10 and cultivation on LB plates overnight for kanamycin-resistant positive clone screening. Sequencing verification confirmed successful construction.

Subsequently, we performed circular PCR via primer design to replace the constitutive promoter J23117 upstream of the repressor gene PcaV with J23101 and J23105 (strength: J23101＞J23105＞J23117). Similarly, plasmid construction was verified by kanamycin screening and sequencing, ultimately obtaining three PCA-responsive operon verification plasmids.

Figure 1. Construction of expression plasmids for the PCA-responsive operon. A. Results of electrophoretic bands of GFP (881 bp) and the pSB3K3 plasmid backbone harboring the PCA-responsive operon (~3.3 kb) after PCR. B. Plasmid map of pSB3K3-J23105-PcaV-P_LV-GFP. C. Sequencing results of target plasmids harboring various constitutive promoters.

(2)Characterization and optimization of expression plasmids for the PCA-responsive operon

We expressed the aforementioned plasmids in E. coli TOP10. Taking the plasmid with J23105 as an example, we conducted a preliminary experiment using PCA induction concentrations of 0, 250 μM, and 400 μM, as selected based on relevant literature. Inducer was added when the OD₆₀₀ value of the bacterial culture reached 0.02. Samples were taken at different time points. After washing cell pellets with 1×PBS, fluorescence intensity (excitation: 480 nm, emission: 520 nm) and OD₆₀₀ values were measured using a microplate reader. The fluorescence intensity per unit cell was calculated to determine the optimal induction time. Results showed that the induction effect of PCA peaked at approximately 3 h, and the leakage expression of the PCA operon in the absence of PCA was not significant. In subsequent experiments, we chose 4 h as the induction time to ensure sufficient growth of the strain, facilitating subsequent centrifugation, precipitation, and washing operations.

After that, we measured the expression levels of PCA-responsive operons regulated by different constitutive promoters under the condition of 4 h induction time. We found that as PCA concentration increased, the fluorescence intensity per unit gradually rose and then reached saturation. Additionally, higher expression levels of the repressor protein PcaV resulted in lower leakage expression. However, this was accompanied by reduced sensitivity to changes in protocatechuic acid concentration. For this reason, we subsequently selected J23105 as the constitutive promoter for PcaV, as it achieves a high signal at the relatively low concentration of 400 μM PCA.

Figure 2. Characterization of the PCA-responsive operon. A. Preliminary experiment on PCA induction time of the operon regulated by promoter J23105 over 0-9 hours. B. Measurement of PCA-induced fluorescence intensity with constitutive promoters of various strengths at 4-hour induction time.

(3)Construction of expression plasmids for the BS-responsive operon

After retrieving the sequence of the bile salt-sensing device from the iGEM Parts Registry, we commissioned a company to synthesize this sequence and constructed its expression plasmid by referring to the aforementioned method for constructing PCA-responsive operon plasmids. Given that the BS-sensing device relies on a transcription factor activation mechanism, we selected the strong constitutive promoter J23101 as the upstream promoter for the transcription factor ramA. Meanwhile, we adjusted the ribosome binding site (RBS) sequences upstream of GFP including RBS30 and RBS32) to regulate GFP expression levels and reduce its leakage expression. Similarly, we first amplified the target fragments by PCR, verified the bands via electrophoresis, then constructed the plasmids by transforming the Gibson-assembled product into E. coli TOP10. Subsequently, the transformed cells were cultured overnight on Kan^R LB agar plates, followed by screening of positive clones and sequencing verification.

Figure 3. Construction of expression plasmids for the BS-responsive operon. A. Results of electrophoretic bands of pSB3K3-J23101-RamA-P_acrRA-GFP (~5 kb) with various RBS combinations after PCR. B. Plasmid map of pSB3K3-J23101-RamA-P_acrRA-GFP. C. Sequencing results of target plasmids harboring various RBS combinations.

(4)Characterization and optimization of expression plasmids for the BS-responsive operon

Since the cholate operon primarily functions as a "localizer" for GLP-1 synthesis in the intestine, and there is a certain minimum threshold for intestinal bile salt concentration, its induction time can be considered nearly infinite. Thus, the constructed BS-sensing device was characterized using a 4-hour induction time, the same as the PCA-responsive operon.

We measured the successfully constructed plasmids following the concentration gradients established by the previous team Dundee 2016. Results indicated that the sodium cholate operon exhibited significant leaky expression. Furthermore, fluorescence intensity showed minimal response to increasing sodium cholate concentrations. We tentatively hypothesize that the acrRA operon may be subject to regulatory factors beyond cholate. Moving forward, we planned to further improve the expression pathway of the cholate operon by optimizing the strain system (such as enhancing heterologous protein tolerance and adjusting the plasmid backbone according to the original work). For subsequent AND-gate circuit assembly, we employed a 4-hour induction period with 25 μM cholate, and the RBS32-33 combination to minimize expression leakage.

Figure 4. Measurement of BS-induced fluorescence intensity with various RBS combinations at 4-hour induction time for the constructed plasmids.

2.Characterization and optimization of the complete AND-gate circuit

After characterizing the two operon elements separately, we combined them with orthogonal intein elements to construct the AND-gate logic. Specifically, we cloned the two operons, coupled split inteins, and split transcription factors into the pSB3K3 plasmid, while cloning the P₁₆ promoter and GFP into another pSB4A3 plasmid containing the ampicillin resistance. Subsequently, these two plasmids were co-transformed into E. coli TOP10 to characterize the function of the AND-gate.

(1)Construction of expression plasmids for the AND-gate circuit

We first obtained the plasmid containing orthogonal intein-transcription factor elements from Prof. Wang's lab. Subsequently, we amplified the PCA-responsive operon, N-terminal and C-terminal halves of the split intein-transcription factor, cholate operon, and pSB3K3 plasmid backbone separately via PCR, followed by Gibson Assembly to obtain the complete upstream AND-gate plasmid. Next, we designed primers with homologous arms to assemble and integrate the P₁₆ promoter, GFP, and pSB4A3 plasmid backbone, constructing the downstream reporter plasmid. All the aforementioned plasmids were transformed into TOP10 competent cells, with positive clones screened on LB plates containing the corresponding antibiotics, and verified by subsequent sequencing.

Figure 5. Construction of the AND-gate plasmids. A. Plasmid map of pSB3K3-J23105-PcaV-P_LV-SspGyrB^C-ECF16^C-J23101-RamA-P_acrRA-SspGyrB^N-ECF16^N. B. Plasmid map of pSB4A3-P₁₆-GFP. C, D. Sequencing results of target AND-gate plasmids.

(2)Characterization and optimization of expression plasmids for the AND-gate circuit

After assembling the two AND-gate plasmids, we co-transformed them into TOP10 competent cells for characterization, later screened and amplified using LB plates containing Kan^R and Amp^R double resistance. First, we fixed the sodium cholate concentration at 25 μM, induced the cells with PCA at gradient concentrations of 0-1000 μM for 10 hours, and determined the unit fluorescence intensity of GFP expressed by the bacteria to verify the function of the AND-gate circuit and explore the optimal PCA induction concentration. The results showed that the unit fluorescence tended to saturate after treatment with 400 μM PCA, so 400 μM PCA was used in subsequent experiments.

Subsequently, we further optimized the induction time to characterize the process of intestinal flora from receiving PCA induction to producing GLP-1. For this purpose, we fixed the concentrations of PCA and sodium cholate at 400 μM and 25 μM, respectively, and set up two control groups: TOP10 strains transfected with empty plasmid pSB3K3 with PCA and sodium cholate added, and TOP10 strains transfected with the AND-gate plasmid but only sodium cholate added, to further verify the effect of AND-gate. The results showed that an approximately 5-fold on-off signal was generated at 5 hours of induction, indicating that the AND-gate logic functions properly with a relatively short overall induction time.

Figure 6. Characterization of the AND-gate genetic circuit. A. Measurement of GFP unit fluorescence intensity with PCA at induction concentrations ranging from 0 to 1000 μM and BS at a fixed concentration of 25 μM for 10 hours. B. Measurement of the unit fluorescence intensity of GFP for verification of the AND-gate logic at 3.5-6.0 h.

3.Summary

We characterized the performance of the two operons separately, then assembled them into complete AND-gate plasmids for functional verification, and initially constructed a “inducer” + “localizer” sensing system. This enables engineered bacteria to produce the downstream GLP-1 upon induction by the green tea metabolite PCA when in the intestine.

In the future, the AND-gate sensing system can be optimized and its applications expanded from multiple dimensions. On the one hand, induction conditions can be further optimized, such as exploring the optimal concentration of PCA in vivo and the combination of induction time to reduce the leakage expression rate of the system, on the other hand, the robustness of the system under different simulated intestinal environment conditions including different pH and microbial interference can be tested to improve its adaptability in complex intestinal environments. In addition, the cholate operon can be further optimized to construct a truly long-acting intestinal localization system.

In conclusion, the intestinal environment sensing system is an extremely complex system, and further improvements can be made based on more newly discovered specific molecules or receptors in the future.

Circuit 2: Amplification & Feedback

1.Characterization and optimization of the Amp30^E elements and GURB3-2 promoter separately

The amplification & feedback circuits consist of an Amp30^E-based amplifier (Part:BBa_K2967000) and an hrpV regulatory element (Part:BBa_K2967006). However, due to time constraints, we only used the PCA operon to replace the upstream AND-gate signal input during actual verification, to test the amplifier performance and preliminarily verify the feedback regulation function.

(1)Construction of expression plasmids for every element of the Amplification & Feedback circuit

First, we commissioned a company to synthesize the PCA operon, which was used to replace the arsenic ion sensor in plasmid pXWJ109AsAmp30E provided by Prof. Wang. Then, through primer design and circular PCR, we replaced the constitutive promoter upstream of the repressor protein PcaV, constructing three amplifier plasmids with different expression intensities. Subsequently, we characterized the glucose uptake-repressive promoter GURB3-2. The GURB3-2 fragment, GFP, and pSB3K3 plasmid backbone were assembled via Gibson Assembly, then transformed into E. coli TOP10 for positive clone screening. Ultimately, we successfully constructed the amplifier plasmids and the GURB3-2 characterization plasmid.

Figure 7. Construction of the Amplification & Feedback plasmids. A. Plasmid map of pSB3K3-J23117-PcaV-P_LV-hrpR-hrpS-P_hrpL-GFP. B. Sequencing results of the amplifier plasmids with various constitutive promoters (J23117, J23105 and J23101). C. Plasmid map of pSB3K3-GURB3-2-GFP. D. Sequencing results of the target feedback plasmid.

(2)Characterization and optimization of expression plasmids for every element of the Amplification & Feedback circuit

We transformed the amplifier plasmid into E. coli TOP10 competent cells for characterization, with the initial inoculation OD₆₀₀ value set to 0.02. A 4-hour cultivation preliminary experiment was conducted within the PCA concentration range of 0-200 μM. Subsequently, we selected 0, 100, 250, and 400 μM PCA to determine the fluorescent amplification fold after replacement of the three constitutive promoters, so as to characterize the amplifier function.

Results showed that below 200 μM PCA, the amplifier could still achieve an amplification effect of nearly two orders of magnitude. Meanwhile, among the three promoter replacement systems, the J23101 promoter group exhibited the lowest amplification leakage, with stable overall amplification fold. The signal intensity without amplification was also consistent with the AND-gate output signal. In contrast, the J23117 promoter group showed significantly higher leakage, generating strong signals even under the condition of 0 μM PCA (with no induction), indicating insufficient stability of the amplifier's amplification effect on high-sensitivity sensors. Thus, the J23101 promoter group is planned to be used as the amplification loop in the construction of the complete Amplification & Feedback circuit in subsequent experiments.

We subsequently characterized the function of the GURB3-2 promoter. The experiment followed a similar procedure as above. We set up a glucose concentration gradient of 0-5 mM to conduct a preliminary fluorescence measurement experiment and later selected glucose concentrations of 0 mM (simulating fasting state), 5 mM, and 10 mM (simulating feeding state), corresponding to intestinal glucose levels under the two physiological states, respectively, for further optimization of induction time parameters.

It turned out that we successfully verified the glucose uptake feedback inhibition regulatory function of the GURB3-2 promoter. Unit fluorescence intensity showed a negative correlation trend with increasing glucose concentration, and the "on-off" effect between fasting and feeding states was significant. During the monitoring period of 2.5-5 h, the fluorescence signal difference between the two states was close to one order of magnitude. Among these time points, the unit fluorescence intensity at 4 h was relatively high and matched the response characteristics of the amplification circuit. Therefore, 4 h was determined as the induction time for the verification of the entire genetic circuit in subsequent experiments.

Figure 8. Characterization of the Amplification & Feedback circuit elements. A. 4-h preliminary experiment for amplifier plasmid expression induced by 0-200 μM PCA. B. Measurement of amplification folds of the amplifiers with different expression levels at 0, 100, 250, 400 μM PCA. C. Characterization of the GURB3-2 promoter under 0–5 mM glucose concentration gradient. D. Functional effect of the glucose concentration feedback regulation circuit under simulated feeding/fasting states.

2.Characterization and optimization of the complete Amplification & Feedback circuit

After characterizing the aforementioned elements, we integrated the two circuits into the pSB3K3 plasmid to verify the amplification and feedback regulation functions of the circuit. Specifically, we first ligated the GURB3-2 promoter with the hrpV gene obtained from Prof. Wang’s lab, then inserted this fragment into the amplifier plasmid harboring the J23101 promoter via Gibson Assembly. After that, we designed an orthogonal experiment for PCA concentration and glucose concentration in TOP10 to investigate the combined effects of amplification and feedback regulation on the expression levels of GFP.

(1)Construction of expression plasmids for the A&F circuit

We obtained the GURB3-2-hrpV fragment and the amplifier plasmid backbone via PCR amplification, then constructed the complete Amplification & Feedback (A&F) plasmid through Gibson Assembly and transformed it into E. coli TOP10, followed by plating on kanamycin-containing LB selective agar plates, and cultured overnight. After screening positive clones and performing sequencing verification, the final plasmid pSB3K3-J23101-PcaV-GURB3-2-hrpV-P_LV-hrpR-hrpS-P_hrpL-GFP was obtained.

Figure 9. Construction of expression plasmids for the Amplification & Feedback circuit. A. Results of electrophoretic bands of GURB3-2-hrpV (663 bp) and the amplifier plasmid vector harboring the constitutive promotor J23101 (~6.5 kb) after PCR. B. Plasmid map of pSB3K3-GURB3-2-hrpV. C. Plasmid map of pSB3K3-J23101-PcaV-GURB3-2-hrpV-P_LV-hrpR-hrpS-P_hrpL-GFP (A&F circuit). D. Sequencing results of the target A&F expression plasmid.

(2)Characterization and optimization of expression plasmids for the A&F circuit

The aforementioned A&F plasmids were transformed into TOP10 competent cells, followed by an orthogonal experiment to determine the effects of glucose concentration and PCA concentration. The initial inoculum OD₆₀₀ was set to 0.02, and after 4 hours of induction, the unit fluorescence intensity was measured to verify the effects of amplification and feedback regulation. Meanwhile, we used TOP10 cells containing the PCA-responsive operon and the amplifier element plasmids as controls to exclude interference from excess glucose on fluorescence intensity in E. coli lacking the feedback regulation circuit.

Results showed that the A&F plasmids could achieve high-level GFP expression with increasing PCA concentration, and the inhibitory effect was further relieved with increasing glucose concentration. Specifically, the feedback regulation circuit could appropriately reduce the synthesis of downstream products and thus alleviate the metabolic burden of the strain when no additional glucose was added (simulated fasting state). Further Pearson correlation analysis revealed no significant association between glucose and fluorescence expression in strains without the feedback regulation circuit. This indicates that the addition of glucose itself in LB liquid medium does not significantly affect the function of the amplification circuit, thereby further confirming the function of feedback regulation.

Figure 10. Characterization of the Amplification & Feedback genetic circuit. A. Results of the orthogonal experiment of glucose and PCA concentrations for cells harboring the A&F plasmid with 4-h induction. B. Pearson correlation analysis heatmap matrix of cells harboring the PCA-responsive operon plasmid. C. Pearson correlation analysis heatmap matrix of cells harboring the amplifier plasmid.

3.Summary

The Amplification & Feedback circuit is mainly designed to address two issues: the low abundance of E. coli in the intestine, and the transmembrane loss of secreted GLP-1 into the bloodstream. Additionally, we aim to precisely regulate GLP-1 synthesis levels according to feeding/fasting states, avoiding excessive metabolic burden on the EcN. However, in actual experiments, we found that feedback regulation is not direct enough and is easily affected by the sensitivity of the AND-gate input signal, resulting in suboptimal regulatory effects. Moreover, the synthesis level of the amplifier under high PCA concentrations is unsatisfactory, which may be related to the bacterial tolerance to high PCA concentrations. Furthermore, the replacement of the upstream AND-gate circuit with the PCA operon is relatively simplistic, and subsequent works need to verify the amplification and feedback regulation capabilities under complex conditions through combined genetic circuits.

In conclusion, we have preliminarily verified the synthesis level regulatory function of the A&F circuit. It is hoped that subsequent optimization of the expression circuit and refinement of induction parameters in the human body will provide references for the actual construction of the circuit, truly achieving intelligent regulation of probiotic synthesis in response to feeding/fasting states.

Conclusions

Through the construction of genetic circuit plasmids and optimization of expression conditions, we have preliminarily clarified the performance and characteristics of each elements. In the future, we will further explore the construction of complex sensing circuits based on this, to ensure that GLP-1 synthesis is intelligently controllable and can be "sufficiently produced" for hypoglycemic and anti-obesity purposes, while exploring more quantitative circuit designs.