Experiment

1.Verifying the feasibility of the sensory module

The feasibility of our sensory module was verified - the increase in butyrate concentration indeed induced an increase in the expression of tagged proteins in the engineered bacteria, i.e., it was verified that our engineered bacteria could indeed be more sensitive to the increase in butyrate concentration.

2.Reverse thinking to verify the feasibility of the sensory module

The feasibility of our sensory module was verified - a rise in butyrate concentration did induce an increase in the expression of the engineered bacterial tagged protein Cl, i.e., it was verified that our engineered bacterium was indeed more sensitive to the rise in butyrate concentration.

3.Validating the feasibility of metabolic module

Using untreated pET28a as a control group, we performed experiments such as WB, fluorescence intensity (OD600) measurement, and gray value analysis to verify the feasibility of the metabolic module.

4.Validating the feasibility of malic acid kill-switch circuit

Malic acid kill-switch is for in-vivo urgent containment, which means the patient can control the process as their wish by take in some malic acid with good flavor.

Pre-experimentation via zone of inhibition showed that malic acid addition can inhibit the bacteria growth. Then we carried out OD evaluation, which is unsatisfactory for OD evaluation cannot distinct living and dead individuals well. So we performed interval plate spreading at 2h, 4h, 6h, 8h, 10h, 12h under different concentrations of malic acid co-culture, which showed that malic acid addition can exert killing effect.

Note: Since our sequence has a high number of reverse repeat sequences and the routes involving the toxin proteins are likely to kill the bacteria outright due to short circuit, we encountered numerous difficulties in constructing and extracting this plasmid. Future teams should carefully consult the sequence and construct the plasmid when designing similar kill switch circuit.

5.Validating the feasibility of rhamnose kill-switch circuit

Rhamnose kill-switch is for environment protection, that is to say, when bacteria is excreted outside and rhamnose is diluted, bacteria will die due to the kill-switch, which is triggered by low concentration of rhamnose.

Pre-experimentation via plate spreading showed that rhamnose addition or not has a significant effect on the survival of bacterial and subsequent OD evaluation under different concentration of rhamnose co-culture for 36 hrs showed that groups with higher concentration of rhamnose grew better, which met our expectations.

Although our results are largely as expected. However, in our results, no matter in the plate spreading or the growth curves, it can be observed that some of the engineered bacteria could not be killed despite the absence of rhamnose. This is probably due to the insufficient expression of mazF in some individuals, and in the future, attempts could be made to replace the toxin proteins or introduce an amplifier to make up for this deficiency. Meanwhile, it should be noted that since the OD curves could not distinguish the survival state of the bacteria, the downward trend could not be demonstrated, even though the bacteria were in a state of mass apoptosis in this period of time.

6.Validating the feasibility of tannin and mucin encapsulation

We produced a layer-by-layer encapsulation with tannin and mucin for bacteria and verified its ability to resist gastric juices by using SGF (simulated gastric juices).

The results showed that the encapsulation only had an acceptable impact on the growth viability of Escherichia coli and exhibited significant protective effects when co-cultured with SGF. Therefore, the encapsulation scheme aligns closely with our expectations. However, encapsulated bacteria also suffer a lot by SGF, which means our protocol and process needs further refinement by paying attention to some details in the referred study.

Model

Model	Method or Equation	Results
Host strain selection	bidirectional Mendelian randomization	E.coli was a safe and efficient host strain for our engineered bacteria.
E. coli growth		Well fitted for prediction of E. coli intestinal concentrations.
Product production		Well fitted for prediction of 5-HT intestinal concentrations.
Kinetic model of drug elimination		Useful for personalizing dosing schedules and calculating steady-state concentrations in the gut.
Elimination model of E. coli		Gained the crucial parameter for prediction of E. coli elimination.

Product design and future plans

Step1 Optimize and test quorum sensing module

In the course of the project, we hypothesized that the amount of engineered bacteria can be automatically controlled by the quorum sensing system of the bacteria themselves, and that the amount of engineered bacteria can be automatically maintained at the proper amount for a long time, thus performing the preventive, monitoring and therapeutic functions of engineered bacteria without manual control. We will then improve and test our quorum sensing module.

Step2 Improve the sensitivity of current modules

In order to maintain the delicate balance of healing and intestinal homeostasis, the sensitivity of the sensory module, the efficiency of the metabolic module, and the stability of the quorum sensing module still require further improvements, calling for more detailed exploration in the future.

Step3 Integrate modules into acid-tolerant strains

Due to the defective nature of the E. coli system, we intend to conduct further experiments in the future using suitable acidophilic strains (e.g. Lactobacillus acidophilus) as chassis bacteria.

Step4 Find a more suitable way for transportation and restoration

We envisioned that the engineered bacteria we constructed would be added to foods such as yogurt. When people consumed these foods, the engineered bacteria would colonize their intestines and carry out their work. But how to maintain the survival of the engineered bacteria after transportation, restoration and consumption before they arrived at the intestines still needs to be explored in more detail.

Step5 Carry out experiments on animals

Application of a medical product must be tested in animals. Due to conditions and time constraints, we were unable to conduct in vivo validation experiments. Subsequently, we plan to conduct more tests using acid-resistant bacteria and carry out in vivo experiments to validate the therapeutic effect in constipated mice model,to obtain more convincing experimental data in vivo.

The envisioned process of treatment

Once the genetic machine has been successfully designed, we are ready to conduct further experiments in the future using acidophilus as the chassis bacteria due to the defective nature of the E. coli system. We expect to add engineered bacteria to foods such as yogurt, and when people consume these foods, our engineered bacteria will enhance their competitiveness in the intestines with the help of the quorum sensing module until they reach an appropriate population density. When possible constipation occurs, the engineered bacteria will sense the decreasing butyrate level and secrete serotonin to regulate intestinal function, thus promoting intestinal peristalsis, enhancing intestinal secretion, and finally relieving constipation. At the same time, when the bacteria are expelled along with the fecal, we do not need to worry about contaminating the environment because once the engineered bacteria are out of the environment at 37°C, the sophisticated kill switch circuit will exterminate them in time.