With the goal of better characterizing the principles of biobricks in diagnosing and treating inflammatory bowel disease (IBD) that we have used in the system, and to ensure the security of our system, we designed four experiment cycles under the guidance of DBSL rules to study our system's practicability. The goals of the three groups of engineering modifications are:

1. To study the sensitivity of the IBD detecting system,

2. To test how the signal amplifying system works,

3. To verify the complete treatment system,

4. To examine the system’s safety by detecting the suicide switch.

In order to characterize the sensitivity of the IBD detecting system controlled by two key molecules ThsS and ThsR, we decided to design a signal-reacting system controlled by ThsS and ThsR and place it in an environment with thiosulfate--an important biomarker for inflammatory bowel disease. We will detect and study the system's response to different concentrations of thiosulfate.

We added ThsS and ThsR biobricks into the pSB1C3 plasmid and named it as pSB1C3-ThsS/R. After the ThsS and ThsR components, we added a sfGFP tag to detect the responsiveness of the IBD detecting system.

We transformed the plasmid into the chassis organism, Nissle 1917, and induced it with thiosulfate solutions of different concentration gradients. Later,we detected the fluorescence intensity by the microplate reader.

We found out that our IBD detecting system can diagnose IBD sensitively.

Considering levels of many disease-related biomarkers are dynamic and difficult to capture and monitor in native environments, we decided to add a CRISPR-based genome(CBE) editing system to amplify the inflammatory signal reaction.

We added a cytosine base editor, TadA-CDd V106W into the pSB1C3-ThsS/R plasmid and named it as pSB1C3- TadA-CDd V106W. And in this added system, the promoter of sfGFP was replaced with a CRISPR-editable ACG-tag sequence, which silenced its translation, TadA-CDd V106W was expressed under the control of ThsS/R to respond to thiosulfate levels in environment.

We transformed the plasmid into the chassis organism, Nissle 1917, and induced it with thiosulfate solutions of different concentration gradients. Later, we detected the fluorescence intensity with the microplate reader.

The newly added CRISPR-based editing system works out well under stimulated IBD environments.

With the goal of verifying the complete treatment system, based on the first two cycles of experiments, we have also incorporated drug proteins and their secretion system. As our drug protein, AvCystatin plays a crucial role in regulating macrophage immune responses. It can activate cellular MAP kinase and induce the expression of IL-10 and IL-12/23p40, thereby effectively alleviating gut inflammation.

Based on previously built systems, we have built up the complete treatment systems. And because the plasmid will be too big if we put the drug protein and its secretion system into the same plasmid, we divided our therapeutic system into two plasmids that could work together, with AvCystatin, the drug protein in the first plasmid pSB1C3-TadA-CDd V106W and HlyB, HlyD, the secretion system into plasmid pET-9a.

Plasmid pSB1C3-TadA-CDd V106W added with AvCystatin was named pSB1C3-AvCystatin, plasmid pET-9a carried HlyB and HlyD system was named pET-9a-Hly

We transformed the plasmid into the chassis organism, Nissle 1917, and induced it with thiosulfate solution from the optimal response concentration in the previous cycles. Later, we assessed the expression of drug proteins and the synergistic effect of the dual plasmid system using by western-blot.

The complete IBD treatment system works can diagnose and treat IBD effectively.

In order to ensure the system’s safety by controlling drug expression, we designed a suicide switch and put it into the IBD treatment system. The key molecule in the suicide activating part is PnorV promoter and the bacterial enhancer binding protein NorR, which could be inhibited when the level of another promising IBD detecting biomarker--nitric oxide decreased. This leads to the inhibited expression of the CI protein. Thus, the PL promoter is derepressed, inducing the expression of the MazF toxin protein, which will lead to cell apoptosis. Moreover, studies have shown that MazF has no toxin to healthy human cells so it can be used in vivo.

Similarly, considering the plasmid size and the synergistic effects of different systems, we chose to add the suicide switch to the pET-9a-Hly plasmid. We sequentially added the PnorV promoter, the NorR binding protein, the CI protein, the PL promoter, and the MazF protein after the HlyD component. The newly developed plasmid was named pET-9a-suicide.

We transformed the plasmid into the chassis organism, Nissle 1917, and induced it with thiosulfate solution from the optimal response concentration in the previous cycles. All experiments were conducted in a nitrogen monoxide environment to simulate the intestinal environment of patients with IBD. We then assessed the expression of drug proteins and the synergistic effect of the dual plasmid system using by western-blot. Later, we removed the DETA-NO solution used to provide nitric oxide to simulate the intestinal environment after the improvement of IBD, and after removing the solution, we collected colonies onto slides at 1 hour, 2 hours, and 3 hours, performed trypan blue staining, and observed under the microscope to count dead cells in order to evaluate the efficacy of the suicide system.

We found that the suicide system can effectively respond to and eliminate chassis cells in a non-IBD environment, thereby preventing drug overexpression and ensuring the safety of the system.