The goal of our project is to alleviate allergy symptoms caused by cats using the engineered E. coli Nissle 1917 which can neutralize antigen to colonize in cat mouth. Until now, we have confirmed the feasibility of our project in E. coli DH5α and BL21(DE3). As is described in our design page, our project has three core modules and a suicide module. The "Colonization" module and the "Blocking" module are connected by an NOT-Gate, which allow engineered bacteria to survive in the cat mouth for a long time and better achieve the prevention of allergy to cat. The "Sterilization" module ensure the health of cat mouth. The final product is a probiotic nutritional supplement.

In order to prove our concept, we have conducted many experiments in the last 3 months after entering the lab. Most of the core modules of our project have been validated so far. However, due to time and equipment constraints, our project has not yet been validated on rats. With the preliminary experimental results available so far, we have determined that our project is practical and feasible for further optimization and extension in the future.

The primary experiments were implemented in common E. coli strain DH5α or BL21(DE3). The "Colonization" module can produce CsgA-Hsa which help engineered bacteria adhere to tooth. Then the "Blocking" module can express two scFvs which can bind antigens and prevent IgE engagement. Moreover, The "Sterilization" module can function better to prevent S. mutans from growing by expressing ClyR. Around these core components, we have demonstrated our engineered bacteria through experiments and other methods.

Colonization

In order to ensure that the engineered bacteria can stay in the cat's oral cavity for a period of time to fulfill its role, our first concern is the bacterial colonization ability. After literature references, we chose csgA associated with biofilm from E. coli K-12 and hsa associated with adhesion protein from Streptococcus gordonii str.Challis. CsgA is a Curli amyloid fibrils protein secreted extracellularly to mediate host cell adhesion and contributes to biofilm formation, which promotes bacterial resistance to environmental stressors. Hsa anchors to the cell wall and binds to salivary acids in the cat's mouth, helping our bacteria to adhere to the salivary pellicle.

To balance the biofilm forming ability of CsgA and the salivary acid adhesion ability of Hsa, we designed the CsgA-Hsa fusion protein, the specific information of this component is at (http://parts.igem.org/Part: BBa_K4645003). Theoretically, the engineered bacteria secrete the fusion protein extracellularly, the amyloid fibrillar protein CsgA portion of the fusion protein accumulates around the bacteria to form a biofilm, and Hsa firmly binds the salivary acid molecule. The Hsa is divided into five regions, NR1, SR1, NR2, SR2, and CWAD, with NR2 being the main salivary acid-binding site, SR2 acting as a molecular stalk and CWAD is the cell wall anchoring region [1]. We intercepted the NR2 region of Hsa and linked it to the C-terminus of CsgA with the linker of (GGGGS)₄ [2].

Using different concentrations of IPTG to induce E. coli BL21(DE3) containing the target gene, and then incubate the target protein with cat tooth to detect the fluorescence intensity of cat tooth [3].The results showed that the fluorescence intensity of the experimental group added with 0.1 mM IPTG and 1 mM IPTG was significantly different from the two control groups and other concentration induction groups (Figure 2). We repeated this experiment several times and obtained the same results. From this, it is determined that the fusion protein CsgA-Hsa indeed has an adhesive effect on saliva coated cat tooth.

After E. coli BL21(DE3) was induced with 0.1 mM IPTG, the OD₆₀₀ values of the group induced by IPTG showed significant differences compared to the other two groups [4]. We can determine that the biofilm formation ability of the engineered bacteria containing the target protein is very significantly different from the two control groups at an inducible concentration of 0.1 mM IPTG, which confirms that CsgA-Hsa indeed enhances the formation of bacterial biofilm.

After induction and staining with IPTG with gradients of 0 mM, 0.1 mM, 0.5 mM, and 1 mM, the samples were observed under a fluorescence microscope [5]. Under all concentrations of induction, the biofilm formation effect of 0.1 mM IPTG was the best, with significant differences between other groups, and the biofilm formation effect was inversely proportional to the IPTG concentration between 0.1-1 mM IPTG. The results of this experiment are consistent with those of Cat dental imprinting, and both are optimal under conditions induced by 0.1 mM IPTG.

Blocking

The function of "Blocking" module is to produce and secrete scFvs (NeuA amd NeuB) to bind Fel d 1 and prevent IgE from people who are allergic to cats engagement. Then preventing allergy reaction [6,7].

To test this module, firstly we need to purify FelD expressed by E. coli BL21(DE3) and verify it can bind to IgE from people who are allergic to cats. By SDS-PAGE analysis we identified the expression of FelD which can be seen in lane 2 (Figure 5, A). To verify the ability of FelD bind to IgE from people who are allergic to cats, FelD was coated and IgE from allergic human donor sera or strandard IgE was incubated at 37℃ for 90 min. Then we Added biotin-labeled IgE antibody and HRP-conjugated Streptavidin. Finally we measured absorbance at 450 nm. Absorbance value present the effect of FelD bind to IgE. So the result of ELISA verified that our FelD has the ability to bind IgE from people who are allergic to cats (Figure 6). Additionally, we also tested the binding of IgE to different concentrations of FelD.

Subsequently, we verified whether NeuA, NeuB, and the combination of NeuA and NeuB had a blocking effect on FelD and IgE from people who are allergic to cats. Firstly, the result of SDS-PAGE analysis determined that NeuA and NeuB were expressed and purified and we can see them in lane and lane (Figure 5, B). Then through blocking ELISA, we found that the blocking effect of NeuA and NeuB on FelD is limited when they are used alone, and the blocking effect is very obvious when NeuA and NeuB are combined to FelD (Figure 7). The specific scheme of this blocking ELISA is to coated FelD on Microtiter plates and incubate different scFvs combinations at 37℃. The IgE from allergic human donor sera IgE from allergic human donor sera and then biotin-labeled IgE antibody and HRP-conjugated Streptavidin were added successively. Finally we measured absorbance at 450 nm. Absorbance value present the effect of FelD bind to IgE. Therefore, we confirm "Blocking" module can work.

QS system

Vibrio cholerae uses a quorum-sensing (QS) system composed of the autoinducer 3,5-dimethylpyrazin- 2-ol (DPO) and receptor VqmA, which together bind to Promoter qtip and activate transcription [8]. According to reports in the literature, we found that E. coli itself can produce DPO [9], and the concentration of DPO is positively correlated with the amount of E. coli. We made use of this QS system to realize regulation of downstream circuit. To make sure this system could work as expected, we built this circuit (Figure 8) and tramsformed into E.coli.

Firstly, we need to make that VqmA_phage could be expressed in E.coli. By SDS-PAGE analysis we identified the expression of VqmA_phage.

We inserted eCFP reporter gene behind qitp promoter (Figure 10) to verify whether qtip promoter could work as expected in E. coli BL21(DE3). However, bacteria that have transformed this plasmid never shown any fluoresce, even though the sequence didn’t mutate. Later, we found that the sequence of qtip promoter contains more than one initiation codon. This may lead to the losing of efficiency of ECFP. So, we tried to delete sequence between eCFP and the last initiation codon and between eCFP and the penult one. Sad to say, we were not able to make this part work finally.

So, we implemented the alternative plan: AHL quorum-sensing system. However, compared to the originally designed system, it lacks the same level of specificity. Thanks ShanghaiTech-China for offering BBa_K4115039, BBa_K4115040 to us. So, in the construction of Not-Gate, we use AHL quorum-sensing system to realize regulation.

NOT-Gate

When the number of engineered bacteria is insufficient, bacteria will express Hsa-CsgA, which is in the downstream of TetA promoter. With the growth of bacteria, AHL accumulates. Then LuxR will combine to Lux promoter and activate transcription of TetR, NeuA and NeuB. TetR will represses the expression of Hsa-CsgA.

To verify whether NOt-Gate could work as expected, we built this circuit (Figure 12).

Blank E.coli BL21(DE3), engineered E.coli BL21(DE3) with this biobrick were cultured in microplate reader 37°C, 220 rpm for 10 hours and detected OD₆₀₀, fluorescence intensity (458 nm excitation light, 489 nm emission light) every 10 minutes.

The result shows that in the early growth stage, transformed bacteria expressed Amcyan fluorescent protein continually. When the group density reached the threshold value, the expression of Amcyan fluorescent protein began to be blocked up.

This means that the Not-Gate we built worked successfully.

We learned that Streptococcus mutans has been recognized as one of the principle causative agents of dental caries [10]. The purpose of this module is to prevent cariogenic S. mutans from utilizing the biofilms formed by engineered bacteria on tooth.

We use the chimeric lysin (ClyR), which is the first lysin that demonstrates activity against the dominant dental caries-causing pathogen [11]. The circuit diagram of the module is presented below.

The E.coli BL21(DE3) strain containing plasmid pET28a-ClyR, originated from Hubei Jianxia Laboratory. E. coli strain was cultured to an optical density (OD₆₀₀) of ~0.6, induced by adding IPTG to 0.2 mM, and allowed to grow overnight at 16℃. Purification was performed following the instructions of Ni²⁺-affinity chromatography. To allow ClyR to be secreted out, the protein using the OmpA signal peptide was purified following the same procedure. The following is the purification result of the two proteins.

Next, to determine the dose dependence and time dependence of ClyR's lytic activity, S. mutans UA159 was resuspended to an optical density (OD₆₀₀) of ~0.8 using PBS buffer. In a 96-well plate, we sequentially added 35 μL of ClyR solution at different concentrations and 165 μl of bacterial suspension. The OD₆₀₀ was continuously measured using a Synergy H1 microplate reader under constant shaking at 37℃ for 1 hour. The experimental results are shown in the figure below.

As shown in the above figure, ClyR demonstrates a certain level of antibacterial activity, which becomes increasingly significant as its concentration increases.

In order to dynamically regulate the expression of ClyR in response to environmental changes, we aimed to introduce the quorum sensing two-component system from S. mutans into the engineered bacteria. When the concentration of CSP produced by S. mutans reaches a certain threshold, it activates the nlmC promoter, thereby initiating downstream ClyR expression [12].

To ensure the safety of engineered bacteria, we designed a suicide module to kill the bacterial strains both in the external environment and in a cat's digestive tract, avoiding potential leakage of engineered bacteria.

The suicide circuit contains three main gene pathways: temperature-sensitive pathway, pH-sensitive pathway, and toxin-antitoxin pathway.

Temperature-sensitive suicide

The temperature-sensitive pathway can detect temperature changes. When engineered bacteria leak out into the external environment, the temperature-sensitive pathway takes effect, expressing the toxin protein RelE while expressing CI protein to shut down the expression of the antitoxin protein MntA. The circuit design is shown in the diagram. We performed targeted validation of the key elements contained in the module.

1. Functional validation of RNA thermosensor

To validate the function of the RNA thermosensor, we used two methods to experimentally verify it. In Method 1, we placed the Amcyan protein after the RNA thermosensor in the PUC57 plasmid and expressed the plasmid in E.coli DH5α. After culturing for 4 hours continuously at 36°C, 26°C and 16°C respectively, we detect the relative fluorescence intensity.

In order to examine the possible effects of RNA leakage at 36°C on toxin protein expression and bacterial growth, In Method 2, We referred to the verification method of iGEM21_HZAU-China BBa_K3733043 to verify the RNA thermosensor. For more information you can see here: BBa_K3733011.

The experimental results are shown in Figure 20 and Figure 21.

Both verification methods successfully validated that the RNA thermosensor can function and express proteins at 26°C or lower temperatures. However, the experimental results also showed that there was some leakage from the RNA thermosensor at 36°C, which may lead to some adverse effects.

2. Functional validation of RelE toxin protein

In our experiment, we constructed a plasmid vector with RelE toxin protein connected after the lactose promoter and introduced it into E.coli BL21(DE3). IPTG was used to induce expression and OD₆₀₀ was detected continuously for 4 hours. Samples were taken every 30 minutes, diluted and plated to determine viable cell counts.

Since RelE is a weak toxin protein, as shown in Figure 22, the OD₆₀₀ detection showed obvious inhibition of bacterial growth after induced expression, which delayed the log phase, but the bacteria could still grow in the later phase. The viable cell count method also produced significant results. As shown in Figure 23, viable cell counts decreased rapidly within one hour after induction, exhibiting significant growth inhibition, which demonstrated the bacteriostatic effect of this toxin.

Acid-sensitive suicide and Toxin-antitoxin system

The pH-sensitive circuit can detect pH changes when engineered bacteria enter a cat's digestive tract and take effect, expressing CI protein to inhibit the expression of antitoxin protein MntA. At the same time, it expresses the two-component system RstAB. RstB can sense the decrease in pH to phosphorylate RstA, and RstA can increase the activity of the acid-induced promoter Pasr [13], thus quickly increasing the expression of CI protein.

The toxin protein HepT and antitoxin protein MntA form a toxin-antitoxin pathway, where the expression of MntA protein is inhibited by CI protein.The circuit design is shown in the diagram below. We performed targeted validation of the key elements contained in the module.

1. Charaterization of asr promoter with fluorescence intensity measurement

To validate the promoter activity and its activation range in response to pH, we connected Amcyan protein after this promoter and introduced it into E. coli BL21(DE3). For more information you can see here: BBa K1231000. The experimental results are shown below.

The experimental results are shown in the Figure 27. and Figure 28

As shown in Figure 25, Pasr has almost no expression under pH 7-8 conditions, but begins low-dose expression at pH 6 and gradually increases as the pH decreases, reaching a peak at pH = 5, then maintaining a relatively low expression level as the pH continues to decline. As shown in Figure 26, we selected the final values for differential analysis, which showed extremely significant differences between pH 6, pH 5 and the control group. This experiment validated that this promoter has almost no expression under pH 7-8 conditions, consistent with our project design expectations. Pasr can be used to activate the suicide circuit in the gastric environment of cats to cause engineered bacteria death, thus avoiding potential hazards from leakage.

2. Functional validation of HepT toxin protein

We referred to the method of verifying Hept by iGEM21_HZAU-China BBa_K3733010. To verify the cytotoxicity of HepT, we connected the HepT toxin protein after the lactose promoter and transferred it to E. coli BL21(DE3) using pET-28a(+) vector.

As shown in Figure 27, the toxin protein HepT has a very obvious bactericidal effect, and this effect is dose-dependent, correlating with the induction concentration of IPTG within a certain range.

3. HepT-MntA toxin-antitoxin-CI-PR System

We constructed a circuit to validate the function of the toxin-antitoxin system (HepT/MntA) and the CI-PR system.We constructed the CI protein downstream of the LacI promoter, and constitutively expressed HepT, and connected the antitoxin protein MntA downstream of a promoter repressed by the CI protein, using the pet28a plasmid transformed into E. coli BL21(DE3).

Since the toxin protein HepT is highly toxic, and a strong promoter J23119 and strong RBS B0034 were used, if MntA cannot function, the bacteria transformed with this plasmid should grow slowly or even not survive. But as shown in Figure 29., the bacteria grew well on the plates.We also cultured the E. coli strain to OD₆₀₀ = 0.4-0.6, induced with 1.0mM IPTG, and allowed continuous growth for 4 hours at 37°C. In 96-well plates, by comparing the OD₆₀₀ between the experimental group and control group using the Synergy H1 enzyme-labeled tester, it displayed the function of the antitoxin protein MntA. The experimental results are shown in Figure 30..

Through analysis of the experimental results, we can find that MntA plays an important role in maintaining the survival of bacteria with constitutive expression of HepT. We also successfully verified the availability of the CI-PR (PCI) system. We also found the bacteria in the control group grew slightly slower than normal bacteria, which may be due to low-dose leakage from the LacI promoter.

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