Design

Overview

Our project mainly has three modules of design, namely degradation, inhibition and alleviation. Theophylline as a sensor substance that can specifically turn on the switch of the engineered bacteria in patients. This avoids the expression degradation and inhibition modules when we only need to express alleviation module.

The Schematic diagram of functional overview of our engineered bacteria is shown below (Figure 1).

Figure 1. A Schematic diagram of functional overview of our engineered bacteria.

In the presence of theophylline molecule in the patients' intestine, the functions of the degradation module and the inhibition module are activated, that is, the expression of trimethylamine dehydrogenase, dimethylamine dehydrogenase and formaldehyde dehydrogenase is turned on, so as to accelerate the degradation of TMA and reduce the concentration of this substance.

At the same time, the engineered bacteria can express L-amino acid ligases [1], producing active small peptide inhibitors to inhibit TMA, which was predicted by Model simulation.

In the absence of this signaling molecule, the degradation module switch off. In the meantime, the alleviation module expresses butyric acid, which is beneficial to blood vessels.

However, with or without theophylline molecules, nattokinase, which has a strong protective effect on the composition of blood vessels, is expressed in the alleviation module[2].

Modules

Degradation Module

In this module, we designed the expression of trimethylamine dehydrogenase (TMADH) and dimethylamine dehydrogenase (DMADH) to gradually metabolize the excessive trimethylamine (TMA) in the gut into monomethylamine (MMA). At the same time, even if trimethylamine is degraded by trimethylamine dehydrogenase and dimethylamine dehydrogenase, the by-products produced in the degradation process are also dangerous. As trimethylamine is degraded, it also sheds a molecule of methanol, which quickly oxidizes to formaldehyde in the body. Using the formaldehyde dehydrogenase operon, the system can reduce the concentration of formaldehyde by gene expression when it senses it, and then convert it into non-toxic formic acid.

The circuits of the schemes and brief mechanism of degradation module is shown below (Figure 2).

Figure 2. The circuits of the schemes and brief mechanism of degradation module.

For the FrmRAB operon, in the absence of formaldehyde, the FrmR protein expressed by the FrmR promoter would inhibit the activity of the FrmR promoter. However, when formaldehyde is produced in the environment, the inhibitory effect of FrmR is relieved, and the joint action of FrmA and FrmB protein decreases the concentration of formaldehyde.

Inhibition Module

In this module, we hope to express a common protein, BacD, in Escherichia coli for the synthesis of oligopeptides of less than 10 amino acids, such as dipeptides [1], where we will rely on the results of numerical modeling for the specific inhibition of CutC.

The circuits of the schemes and brief mechanism of inhibition module is shown below (Figure 3).

Figure 3. The circuits of the schemes and brief mechanism of inhibition module.

Alleviation Module

Butyrate can increase the intestinal barrier function, reduce intestinal permeability, circulatory LPS, and systemic inflammation, including the aortic plaques near the expression of proinflammatory cytokines, and macrophage infiltration of plaques and the macrophage's low-density lipoprotein intake and cholesterol esterification is an early step in etiology of atherosclerosis [3]. In the absence of the theophylline riboswitch, the circuit preferentially expressed thioesterases, which hydrolyze the thioester bond of acyl-S-acyl proteins to catalyze the release of SCFAs, including butyric acid [4]. This reduces the level of atherosclerosis and thus buys time for thrombolysis while reducing the formation of new clots.

The circuits of the schemes and brief mechanism of alleviation module is shown below (Figure 4).

Figure 4. The circuits of the schemes and brief mechanism of alleviation module.

Suicide Module

To ensure the safety of the engineered bacteria , we designed a suicide module, killing the strains, which leak outside the body may contaminating the environment. In this suicide module, we designed a heat-repressible RNA thermometer [5] with a toxin protein followed. The former senses the ambient temperature and plays a role in controlling the switch close and open which influence the follow-up pathway, while the latter is able to express the toxin protein HepT [6], acting as a RNase, which degrades mRNA at the transcriptional level and ultimately leads to cell death. Through such design, we ensure the suicide of the engineered bacteria in vitro, furthest reducing the environmental pollution.

The circuits of the schemes and brief mechanism of Suicide Module is shown below (Figure 5).

Figure 5. The circuits of the schemes and brief mechanism of Suicide module.

Modules Switch

How do we make the transition from module to module?

In order to realize an artificial transition between degradation module and alleviation module, we designed the Cre-loxP system. According to the literature, Cre identifies the reverse repeat sequence at both ends of the loxP site and binds to form a dimer, which then binds to a dimer on another loxP site to form a tetramer [7]. LoxP sites are directional, and the two sites connected by tetramers are parallel in direction. The DNA sequence between the two loxP sites is then cut off by Cre. Next, DNA ligases connect these strands quickly and efficiently. If two loxP sites are on the same DNA strand and in the opposite directions, Cre mediates sequence reversal between loxPs. Inserting a promoter and RBS between the two loxP sites allows us to switch from the alleviation module to the degradation module.

The Schematic representation of the Cre/Loxp recombinase system is shown below (Figure 6).

Figure 6. Schematic representation of the Cre/Loxp recombinase system playing two different roles.

Conclusion

So, here are two different scenarios for our primary functional loop design,The inhibition module isn't shown below, please refer to the engineering page for the detail). If patients take probiotics with theophylline, the cre/loxp recombinase system will induce expression of the trimethylamine, dimethylamine, and formaldehyde dehydrogenase gene clusters, metabolizing trimethylamine to less toxic monomethylamine and converting the byproduct formaldehyde to non-toxic formic acid (Figure 7).

Figure 7. Schematic diagram of the key-functionally development of engineered bacteria in the presence of theophylline molecules.

If patients only take our probiotics, the probiotics will only express short chain fatty acids that are beneficial to the body, such as blood vessel protection and intestinal mucosal repair. At the same time, nattokinase is continuously expressed constitutively, which helps patients with thrombolytic therapy and makes probiotics have basic therapeutic effects (Figure 8).

Figure 8. Schematic diagram of the key-functionally development of engineered bacteria in the absence of theophylline molecules.

Design Reflection

If our product is to be used in the real world, we hope that this design will provide patients with a variety of autonomous treatment options: when used in combination with theophylline tablets, the main goal is to reduce the risk of thrombosis by reducing the amount of TMA. However, if the patient finds that the physical condition has recovered well recently, it can promote the thrombolysis and express some other beneficial substances, which can help the patient's health. After all, it has been pointed out that cardiovascular diseases are often accompanied by hypertension, hyperglycemia, hyperlipidemia and other adverse factors [8].

In fact, we were very excited to learn that tea also contains theophylline. It is well known that drinking tea is a relatively healthy lifestyle and diet [9]. In combination with the work of HP, we found that cardiovascular diseases often occur in middle-aged and elderly people, and such people seem to prefer drinking tea. So we thought, can we use tea instead of theophylline tablets, can our product as a probiotic powder for brewing cold tea, and can we make this treatment model more accessible and interesting for people? However, we finally gave up the idea of making tea bags with probiotic powder and tea. The reason was that after experiments, consulting materials and asking teachers' opinions, we found that the theophylline concentration in tea was too low, which was indeed not enough for our engineering bacteria to play a role. This part of the work can be seen in HP analysis.

Reference

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[8] Powell-Wiley T M, Poirier P, Burke L E, et al. Obesity and cardiovascular disease: a scientific statement from the American Heart Association[J]. Circulation, 2021, 143(21): e984-e1010.

[9] Keller A, Wallace T C. Tea intake and cardiovascular disease: an umbrella review[J]. Annals of Medicine, 2021, 53(1): 929-944.