The core targets of our project will be implemented by engineered E. coli. In order to achieve the effect of early and convenient IBD detection, our project is divided into three main modules: "Detection & Report", "Anti-inflammatory" and "Health-care". At the same time, in order to ensure the safety of the project, multiple sets of "Suicide" modules have designed for our engineered bacteria.
The "Detection & Report" module can detect the occurrence of IBD by sensing the signal of increased concentration of nitrate and thiosulfate, and then report the pathological condition of the intestinal tract by producing an easy-to-smelt odor.
The "Anti-inflammatory" module can kill some bacteria that cause inflammation by producing an anti-inflammatory peptide, thereby playing a certain anti-inflammatory effect. The "Health-care" module can secrete a bacterial blue copper-binding protein, which can prevent inflammation from further worsening by stabilizing a protein expressed by a tumor suppressor gene. The "Suicide" module mainly adopts the strategy of realizing suicide by sensing the difference between the temperature of the intestine and the environment.
We design the "Detection & Report" module as an AND-Gate. When both the concentrations of nitrate and thiosulfate increase significantly, the engineered bacteria will report the occurrence of IBD, and then the "Anti-inflammatory" module will be activated, thus to improve accuracy of the project effectively. Meanwhile, the "Health-care" module works continuously to keep hosts' intestinal homeostasis. When our engineered bacteria accidentally enter the environment, they will feel the temperature drop through a RNA thermometer, which will cause the toxins to work and cause them to die.
Figure 1. The whole brief concept of our engineered bacteria.
Figure 2. The outline of gene circuits in the engineered bacteria.
Detection & Report module
According to reports in the literature, the occurrence of IBD is often accompanied by an increase of the concentration of nitrate and thiosulfate, and it is believed that nitrate and thiosulfate can be used as biomarkers to detect whether IBD occurs . The core component of the "detection" module is an AND-Gate system composed of two TCSs, in which the endogenous NarXL of E. coli can sense the change of nitrate concentration and ThsSR from marine Escherichia can respond to high concentrations of thiosulfate [2,3]. NarX is a membrane-bound histidine kinase (HK) that can automatically phosphorylate upon sensing a high concentration of nitrate. Then, the response regulator (RR) NarL can catalyze the transfer of phosphoryl groups to aspartic acid residues in its receptor domain . Phosphorylated NarL (P-NarL) transforms the allosteric into an active conformation, binds upstream of the promoter PyeaR and activates the transcription of another membrane-bound histidine kinase (HK) ThsS and response regulator (RR) ThsR. Then ThsS will undergo autophosphorylation when it feels high concentration of thiosulfate, and then ThsR will repeat the process similar to NarL and produce phosphorylated ThsR (P-ThsR) that binds upstream of the promoter PphsA to activate the "Report" module. The expression of genes related to the module and the "Anti-inflammatory" module.
The circuit and brief mechanism of AND-Gate are shown below (Figure 3).
Figure 3. The circuit and brief mechanism of AND-Gate
Geosmin is a volatile organic compound which gives a special “earthy” smell. Significantly, it has an exceptionally low detection threshold of the odor of 10-100 ppt . It has not been reported that geosmin do harm to pet’s health . Additionally, we consulted Professor Ding, Doctor of Veterinary Medicine, finding out that this smell likely has no adverse effect on ethology. Thus, we selected it as a report for IBD. When two TCSs are activated, gesomin synthase from Streptomyces coelicolor A3(2) (ScGS) will be expressed and the off-Odor substance is released. At this time, the recognizable smell will alarm owners to realize the abnormality of their pets so that they would not miss the golden age of treatment.
The circuit and brief mechanism of Report module are shown below (Figure 4).
Figure 4. The circuit and brief mechanism of Report module and Anti-inflammatory. module
When IBD breaks out, the composition of gut microbiota will be shifted. Lipopolysaccharides (LPS) function as the antigenic determinants of the bacteria, which could trigger the release of proinflammatory mediators in the host via Toll-like receptor 4 (TLR4) and its downstream physiology pathways [7, 8]. And finally, these biochemical reactions will amplify the illness and lead to the breakout of IBD. Luckily, LL-37, one of the antimicrobial peptides (AMPs), could block this pathway by neutralizing LPS . Besides, AMP-mediated bacterial killing mechanism also targets energy metabolism. This mechanism results in the enhanced production of reactive oxygen species (ROS), causing lethal membrane depolarization under aerobic conditions .
In our circuit, the anti-inflammatory module is under the control of the promoter P phsA 342. When the promoter is activated, LL-37/LTA/HyLα will be expressed and then secreted to the outside of the cell with the guidance of signal peptide. By producing AMPs under the pathological condition, the disordered gut microbiota can be balanced, sparing time for further diagnosis and treatment.
We also focus on better health-care for IBD dogs. If dogs with IBD are not treated in time, long-term inflammation may lead to bowel cancer. Azurin has an anti-cancer effect by stabilizing p53, which is a protein expressed by tumor suppressor gene, thereby preventing inflammation from further deteriorating into cancer .
E. coli Nissle 1917 has a certain anti-cancer effect on cancer cells, so we use it as a chassis to express azurin, which could further enhance the anti-cancer effect .
The circuit and brief mechanism of Health-care module are shown below (Figure 5).
Figure 5. The circuit and brief mechanism of health-care module.
To avoid the potential contamination caused by the leakage of our engineered bacteria into the environment, we put forward several optional schemes of the suicide module.
Considering intestinal temperature of dogs is normally higher than room temperature, we decided to enable the bacteria to kill themselves at low temperatures and only survive at intestinal temperature. To achieve such a function, we utilized RNA thermometers and the HepT toxin (and the MntA antitoxin) to build this module.
We designed two different temperature-based suicide schemes (Figure 6). In the first scheme, the expression of HepT would be inhibited by heat-repressible RNA thermometer in dogs’ guts, whereas it could be constantly expressed in vitro and make the bacteria commit suicide . In another scheme, we imported the HepT/MntA toxin-antitoxin system and kept HepT continually being generated. Instead of controlling HepT directly, we applied heat-inducible RNA thermometer to activate the expression of MntA and neutralize HepT in intestine . Both schemes could perform the same expected suicidal function even if we employed diverse RNA thermometers with opposite effects. Furthermore, the specific suicidal scheme could be adjusted properly by changing the selection of RNA thermometers to tackle different room temperatures in different regions when it comes to practical application.
The circuits of two optional schemes and brief mechanism of Suicide module are shown below (Figure 6).
Figure 6. The circuits of two optional schemes and brief mechanism of Suicide module.
To optimize the effect of suicide module and deal with complicated situations in actual implement, we also recommend combining our temperature-based suicide schemes with oxygen-based suicide modules together to build an OR Gate to make the suicide system more sensitive. We designed an optimized circuit containing oxygen-based conditional judgments shown in Figure 7 and we will verify the availability of the improvement scheme in the future work.
Figure 7. Future consideration for the suicide module.
We highly concerned the safety of our bacteria. For this point, suicide module, physical isolation and hardware would work together in our whole project. More details about our physical isolation and hardware could be seen in Implementation(https://2021.igem.org/Team:HZAU-China/Implementation) and Hardware(https://2021.igem.org/Team:HZAU-China/Hardware).
Considering that we will apply our project as a new method for the early detection of IBD in other mammals in the future. However, the concentration of nitrate and thiosulfate in the intestine during IBD vary from mammal to mammal. To allow our assay module to be adapted to multiple mammals, we implemented a two-component system to sense concentration changes by swapping different promoters and RBS, thus allowing our engineered bacteria to swap thresholds for detecting IBD lesions.
For details, please click the link below: https://2021.igem.org/Team:HZAU-China/Measurement
As the ultimate goal of our project is to benefit a variety of mammals, we are particularly concerned about the safety of our project. As mentioned above, in order to make our suicide module better adapted to complex practical applications, we designed an oxygen-related suicide system after reviewing related papers. It is well known that the environment inside the intestine is close to anaerobic while the external environment is aerobic, so we combine the difference of temperature and oxygen to build a new type of suicide system in our future work (Figure 7) . Theoretically, using such a system could more effectively prevent the leakage of engineered bacteria into the environment, since a change in either of the oxygen and temperature environments would cause suicide of engineered bacteria.
In addition, due to time and experimental constraints, our project did not actually conduct in vivo. In our interview(https://2021.igem.org/Team:HZAU-China/Human_Practices), Mr. Donghai Zhou advised that we can conduct preliminary experiments in rats firstly in our future work. Further experiments will be carried out after the desired results are obtained.
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