Team:HZAU-China/Proof Of Concept

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The goal of our project is to achieve early monitoring of IBD and complete the overall validation in E. coli Nissle 1917. Until now, we have confirmed the feasibility of our project in E. coli DH5α and BL21(DE3), and some of the modules have been achieved to be validated in E. coli Nissle 1917. As is described in our design page, our project has three core modules and a suicide module. The "Detection and Report" module and the "Anti-inflammatory" module are connected by an AND-Gate, which allows our project to respond more accurately to different conditions in intestine and mitigate inflammation. The "Health-care" module is there to prevent the progression of inflammation to cancer. Our final product is edible sodium alginate spheres encapsulated with probiotics, accompanied by hardware "Box" and software, which helps the users to apply our program more conveniently.

In order to prove our concept, we have conducted many experiments in the last 4 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.

Demonstration of engineered bacteria

The primary experiments were implemented in common E.coli strain DH5α or BL21(DE3). In addition, we have also done some experiments in E. coli Nissle 1917, which is widely known as one typical kind of Probiotics. "Detection & Report" module, "Anti-inflammatory" module and "Health-care" module are the core functional modules of our bacteria. The "Detection & Report" module can sense nitrate and thiosulfate signals, and then produce ScGS and deliver the secondary signal to activate the "Anti-inflammatory" module. Then the "Anti-inflammatory" module will express a peptide, which can play a role in relieving inflammation by killing some pro-inflammatory bacteria, such as Salmonella Typhimurium SL1344. Moreover, the "Health-care" module can function better to prevent inflammation from deteriorating to cancer. Around these core components, we have demonstrated our engineered bacteria through experiments and other methods.

The outline of our project is shown below (Figure 1):

Figure 1. The outline of genetic circuit.

“Detection & Report” Module

In our project, the "Detection & Report " module can sense the concentration of biomarkers in intestine. After IBD occurs, the concentration of nitrate and thiosulfate in intestine will surge significantly [1]. Two different two component systems (TCS) are under the regulation of nitrate and thiosulfate respectively. Nitrate senor system NarXL and thiosulfate senor system ThsSR were selected [2,3]. They have similar working mechanism; both of them can activate the downstream gene when the concentration of biomarker is abnormally high. In order to guarantee the accuracy of detection and activation of reporting system, we designed an AND gate. When the concentration of biomarkers is high, the AND gate will be activated. The geosmin synthase will be generated to produce geosmin as a report and a small antimicrobial peptide will be expressed (Figure 2).

Figure 2. The design of “Detection & Report” module.

Nitrate sensor system

The NarXL two component system and P_yeaR promoter can meet our design goal. To prove the effectiveness of our design, neGFP was set under the control of P_yeaR. The plasmid was transferred into E.coli BL21(DE3) (Figure 3). According to the literature, the concentration of nitrate in intestines of rats with IBD is around 600 μM, while in intestines of normal rats is around 20 μM. After induced with nitrate, the RFU/OD₆₀₀ values of our engineered bacteria at 1mM induction is higher than RFU/OD₆₀₀ values of bacteria without induction. However, there is no significant difference between the RFU/OD₆₀₀ values of our engineered bacteria induced at 100 μM concentration with ones of the control group (without inducing). These results indicate that the nitrate sensor will be silenced at the normal condition,, while at the illness condition, the nitrate sensor will be activated (Figure 4).

Figure 3. Demonstration circuit of the nitrate sensor system.

Figure 4. The expression condition of neGFP by activation of P_yeaR after adding different concentration of nitrate. The error bar is mean ± standard error.

Thiosulfate sensor system

As is described above and on the design page, thiosulfate sensor system consists of ThsSR TCS and P_phsA promoter. As one of the key components of AND-Gate system, we tested its sensitivity to different thiosulfate concentrations by transforming the verification circuit (Figure 5) below into E. coli DH5α. Then we used a concentration gradient of thiosulfate to induce the sensor system.

Figure 5. Demonstration circuit of the thiosulfate sensor system.

After induced with thiosulfate, the RFU/OD₆₀₀ value of the bacteria samples added 0.01mM thiosulfate has begun to be distinguished from the lower concentration induction groups. Then we repeated this experiment several times and got the same result. We determined that the threshold value of added thiosulfate is between 0.01 mM and 0.1 mM under the control of J23106a in DH5α (Figure 6). Furthermore, this sensor system has a very clear distinction between low and high concentrations of thiosulfate, which represent normal conditions and pathological conditions respectively. The engineered bacteria can distinguish the concentrations of thiosulfate and produce fluorescent proteins, which confirms the feasibility of this sensor.

Figure 6. The expression condition of neGFP by activation of P_phsA after adding different concentration of thiosulfate. The error bar is mean ± standard error.

AND-Gate

After verifying the two biosensors as effective as we expected, we combined two biosensors acting as an AND Gate. In order to verify the function of an AND Gate, the downstream genes were replaced by neGFP and the circuit of this part is shown below (Figure 7). The plasmid was transformed to E.coli DH5α. After induced by nitrate and thiosulfate for 12 hours , we obtained RFU/OD₆₀₀ data of the engineered bacteria. The result is shown below (Figure 8, 9).

Figure 7. The design of this circuit.

Figure 8. The result of AND Gate after 10 hours inducing by thiosulfate and nitrate. Values represent the mean of three biological replicates.

Figure 9. Performance of AND Gate at various combinations of nitrate and thiosulfate. Color bar indicates RFU/OD₆₀₀.

The performance of the AND Gate indicated that there is a significant difference between RFU/OD₆₀₀ value of media with lesion condition and one with normal condition. And the RFU/OD₆₀₀ value of media at the highest concentration of nitrate and thiosulfate is more different from one without nitrate and thiosulfate. In general, the AND Gate not only operated correctly in response to different combinations of nitrate and thiosulfate but also can activate the downstream gene as we expected.

Through these results, we can verify that the AND Gate can work effectively. It can launch the "Report" module and "Anti-inflammatory" module when nitrate and thiosulfate are both at high concentrations.

“Report” module

Plural methods to report were taken into account aiming to produce more significant signals. SYFP(BBa_K864102), a chromoprotein, was chosen for its bright color and fluorescence. With the expectation of a remarkable change in color or fluorescence, we mixed culture with curry, to simulate engineered bacteria in feces. The presented figures (Figure 10) show a relatively significant difference between SYFP expressing bacteria and the control. However, these were gained when the proportion of concentrated culture and curry reaches 1:1, indicating that there is likely no recognizable change in color or fluorescence when the work comes to animals.

Figure 10. Cultures after concentration mixing with curry of 1:1. Culture in Sample 1 is the E.coli expressing SYFP, while it is WT bacteria of similar OD₆₀₀ in Sample 2 and LB medium in Sample 3. A. Mixture under white light. B. Mixture under UV.

Synthesized by ScGS, geosmin was selected as the report substance in our project. A cytotoxicity test was conducted on the chasis, E.coli Nissle 1917, which shows the off-odor substance is quite safe for the bacteria(Figure 11).

Figure 11. Cytotoxicity test of geosmin at different concentrations on E.coli Nissle 1917.

We transferred pET-28(+)-ScGS(with His-tag) into E.coli BL21(DE3) and verified the expression of the geosmin synthase by Ni²⁺-affinity chromatography and SDS-PAGE. In order to identify the synthesis of geosmin, culture in TB medium containing extra 5% glycerol were first induced with IPTG, following by an overnight cultivation at 18℃ and continuing for next 72h at 25℃. Impressively, a strong and unusual odor was recognized from the medium which is rather similar to the smell of moist soil. To further demonstrate that we successfully produced geosmin in engineered bacteria, we examined our sample via headspace liquid-phase microextraction(HS-LPME) and gas chromatography-mass spectrometry(GC-MS) [4]. The results gained by GC-MS fairly show the existence of geosmin in our culture(Figure 12).

Figure 12. Identification of geosmin by GC-MS. A. Total ion current chromatogram of geosmin standard(the peak of it is marked with an arrow peak 1) and the extracted product(the peak of it is marked with an arrow peak 2), which the retention time is similar to each other. B. Mass spectrum of geosmin standard. D. Mass spectrum of the extracted product.

“Anti- inflammatory” module

The function of "Anti-inflammatory" module is to produce and secrete antimicrobial peptide- LTA to balance the disordered gut microbiota in intestine [5,6]. This module is under the regulation of "Detection & Report" module, which reports the current health condition in intestine. Therefore, the identifications of this peptide were conducted and antimicrobial activities was tested.

Figure 13. Tris-Tricine-SDS-PAGE analysis of LL-37, LTA and HyLα expression.

By Tris-Tricine-SDS-PAGE analysis to separate the peptide with low molecular weight, we inferred that LTA could be successfully expressed in E. coli, as there is a clear band in the corresponding place(Figure 13).

Furthermore, we also designed and conducted the antimicrobial activity assay to validate the effectiveness of the LTA. According to the literature, the LL-37 and its derivatives have broad-antibacterial spectrum, which means it can inhibit the growth of diverse strains. We use gram-negative and intestine-existing bacteria Salmonella Typhimurium SL1344 as an example to verify the effect our AMPs. There was an obvious decrease or a gentle growth trend of OD₆₀₀ when we added the AMPs in the culture, compared with the control group(Figure 14).

Figure 14. OD₆₀₀ -Time curve of Salmonella Typhimurium SL1344 in the presence/absence of LTA. The error bars indicate standard error (SEM) of three independent replications.

Taking the typical enteropathogenic bacteria Salmonella Typhimurium SL1344 as a starting point, we have verified that LTA have the capability to inhibit the growth of bacteria. Thus, our AMPs have promising future to balance the disordering gut microbiota whose LPS will undoubtedly deteriorate intestinal health.

”Health-care” module

"Health-care" module is a continuous activated module which is designed to secrete beneficial substance for dogs. Azurin is synthesised by our engineered bacteria which could stabilize p53 and has an anti-cancer effect [7].

In order to verify the feasibility of our health-care module, we used SDS-PAGE gel to observe the secretion of Azurin (Figure 15).

Figure 15. The SDS-PAGE gel of Azurin secretion verification.

To further demonstrate that Azurin generated from our engineered bacteria could combine with p53 successfully, we mixed Azurin(without 6x His tag) with p53(with 6x His tag) and purified them together by a Ni-NTA column. The SDS-PAGE gel of combination verification is shown below in Figure 16. Two correct stripes can be observed in the lane of the gel, which illustrates these two proteins can combine with each other.

Figure 16. The SDS-PAGE gel of Azurin-p53 combination verification.

Observed from the above results, Azurin can be expressed and secreted in E .coli, and it can combine with p53 successfully. Therefore, we confirm "Health-care" module can work.

”Suicide” module

We attached great importance to the suicide module. It can drive engineered bacteria to commit suicide to prevent potential contamination as the bacteria get out of intestine. We designed two schemes to achieve the suicide module. Circuit in the first scheme consists of a constitutive promoter (J23110), a heat-repressible RNA thermometer and the hepT toxin gene [8,9]. The thermometer can inhibit the expression of HepT at 37 ℃, and it would not have a significant influence when the temperature is below 28 ℃. Circuit in the second scheme consists of a constitutive promoter (J23110), a heat-inducible RNA thermosensor, the mntA antitoxin gene and the constantly expressed hepT toxin gene. Bacteria will be killed at 28 ℃ on account of the HepT toxin, and MntA will be expressed to neutralize the toxicity of HepT at 37 ℃. In practice, the engineered bacteria of both schemes will perform the same suicide function at 28 ℃ and will not be killed at 37 ℃. More details about the design of our suicide module can be seen in Design(https://2021.igem.org/Team:HZAU-China/Design).

To verify the function of this module, we transferred the two circuits to the E.coli DH5α respectively. Meanwhile, we also transformed blank plasmid (only with ori and cmR) into DH5α as the control group. We incubated the bacteria in two schemes at both 37 ℃ and 28 ℃, taking the bacteria with blank plasmid as control. As the Figure 17,18 shows, both media in two schemes shaken at 28 ℃ are more limpid than ones shaken at 37 ℃ in the experimental groups, while in the control group, the media shaken at 28 ℃ are almost as turbid as ones shaken at 37 ℃.

Figure 17. The comparison photo of the experimental group (toxin system) and control group incubated at both 37 ℃ and 28 ℃ for 12 hours. Sch.1 means the experimental group in the first scheme (toxin system). Control means the control group. B. The specific OD₆₀₀ data of the experimental group and control group.

Figure 18. The comparison photo of the experimental group (toxin system) and control group incubated at both 37 ℃ and 28 ℃ for 12 hours. Sch.2 means the experimental group in the second scheme (toxin-antitoxin system). Control means the control group. B. The specific OD₆₀₀ data of the experimental group and control group.

We also plotted the quantitative growth curves at 28 ℃ in two schemes (Figure 19). We got OD₆₀₀ data changing over time by culturing our engineered bacteria and control bacteria in an automatic microplate reader for 12 hours. Compared with controls, the growth of both two types of bacteria was inhibited obviously, and it can be observed that the suicide effects of these two schemes are nearly equivalent.

Figure 19. The quantitative growth curves of engineered bacteria of Scheme 1. in 12 hours at 28 ℃. B. The quantitative growth curves of engineered bacteria of Scheme 2. in 12 hours at 28 ℃.

Project improvement

Although under our current design, the performance of the nitrate sensor system is quite perfect. However, the actual situation will be more complicated, and our current design may not be well applied to reality. For this reason, we have changed the performance of the nitrate sensor system by adjusting the expression of the two-component system and point mutations.

Adjusted the expression of the two-component system

In our circuit design, it is based on the combination of the J23109 promoter and RBS B0034. However, combining literature and Model results (More information you can see here) we discovered that promoters and RBS also affect the performance of nitrate sensor system.

Therefore, we have selected two promoters: J23110, J23100 and two RBS: B0032, B0033. Randomly combine the promoter and RBS. We constructed another eight combinations ignored the combination of J23109- B0034 (Figure 20).

Figure 20. The fold change vaule of different combanition for nitrate sensor system. Fold Change Value = RFU (Nitrate = 100mM) / RFU (Nitare = 1μM).

RiboJ is an insulator between promoter and RBS, which can affect translation of the transcript is via the hairpin at the end of the RiboJ sequence, downstream of the ribozyme. It has been suggested that this appended hairpin aids in exposing the RBS [10]. We added RiboJ to the nitrate sensor system in the hope that when the expression of the two-component system is controlled, the expression level can be changed by introducing RiboJ. So we chose the four combanitaions to insert RiboJ (Figure 21).

Figure 21. The fold change value of different combanitions of nitrate sensor system with the RiboJ. Fold Change Value = RFU (Nitrate = 100mM) / RFU(Nitare = 1μM).

More information you can see in Measurement

Point Mutation

Even if the expression level of the two-component system can be changed, the expression levle has already been determined in a specific curcit. Therefore, without changing the expression levle, altering the performance of the two-component system can be achieved by the point mutations.

We selected the 415 amino acid of NarX for point mutation. By converting cysteine to arginine, we successfully lowered the response threshold of nitrate sensor system. (Figure 22)

Figure 22. The expression condition of neGFP by activation of PyeaR after adding different concentration of nitrate. The error bar is mean ± standard error. A. NarX(C415R) B. NarX(WT).

Here, we conduct a preliminary exploration of the performance of the two-component system. Although what we have done is limited, we hope that the approach of changing the performance of the two-component system through two perspectives can guide more exploratory researchers to continue their exploration.

Reference:

[1] Woo S G, Moon S J, Kim S K, et al. A designed whole-cell biosensor for live diagnosis of gut inflammation through nitrate sensing[J]. Biosensors and Bioelectronics, 2020, 168: 112523.

[2] Archer E J, Robinson A B, Süel G M. Engineered E. coli that detect and respond to gut inflammation through nitric oxide sensing[J]. ACS synthetic biology, 2012, 1(10): 451-457.

[3] Daeffler K N M, Galley J D, Sheth R U, et al. Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation[J]. Molecular systems biology, 2017, 13(4): 923.

[4] Xie Y, He J, Huang J, et al. Determination of 2-methylisoborneol and geosmin produced by Streptomyces sp. and Anabaena PCC7120[J]. Journal of agricultural and food chemistry, 2007, 55(17): 6823-6828.

[5] Zhang L, Wei X, Zhang R, et al. Design and development of a novel peptide for treating intestinal inflammation[J]. Frontiers in immunology, 2019, 10: 1841.

[6] Liu W, Dong S L, Xu F, et al. Effect of intracellular expression of antimicrobial peptide LL-37 on growth of Escherichia coli strain TOP10 under aerobic and anaerobic conditions[J]. Antimicrobial agents and chemotherapy, 2013, 57(10): 4707-4716.

[7] Huang F , Shu Q , Qin Z , et al. Anticancer Actions of Azurin and Its Derived Peptide p28[J]. The Protein Journal, 2020, 39(2).

[8] Hoynes-O'Connor A, Hinman K, Kirchner L, et al. De novo design of heat-repressible RNA thermosensors in E. coli[J]. Nucleic acids research, 2015, 43(12): 6166-6179.

[9] Yao J, Zhen X, Tang K, et al. Novel polyadenylylation-dependent neutralization mechanism of the HEPN/MNT toxin/antitoxin system[J]. Nucleic acids research, 2020, 48(19): 11054-11067.

[10] Clifton K P, Jones E M, Paudel S, et al. The genetic insulator RiboJ increases expression of insulated genes[J]. Journal of biological engineering, 2018, 12(1): 1-6.