1. Overall

The core concept of this project lies in successfully constructing an intelligent engineered probiotic that can accurately perform dual therapeutic functions, “repairing the intestinal barrier” and “inhibiting pathogenic bacteria,” under the drive of specific intestinal environmental signals \(bile salts\), while simultaneously possessing environment-dependent biosafety features. To validate this concept, we followed the iterative "Design-Build-Test-Learn" cycle and developed a stepwise validation strategy.

The validation process is divided into two major stages: first, using molecular biology and biochemical methods, we verified the correct construction and expression of functional modules at the molecular and cellular levels. Second, through in vitro functional experiments, we evaluated the therapeutic effects and safety performance of the fully integrated engineered bacteria. Together, these efforts systematically demonstrate the feasibility of the entire design concept.

2. Plasmid Construction and Expression

The successful construction of plasmids and the expression of target proteins form the foundation for the functional implementation of this project. We verified these aspects through the following experiments:

First, all constructed recombinant plasmids \(such as Psod\-accA, P16090\-OLE1, and P16090\-OLE1\-2AA\-DEFB4A\) were validated using restriction enzyme digestion and sequencing analysis. Agarose gel electrophoresis results showed that digestion products matched the expected fragment sizes perfectly, and sequencing results confirmed that no mutations were present, thus verifying the correctness of plasmid construction at the nucleic acid level.

Next, protein expression was validated using Western Blot analysis. The results showed that, in the presence of bile salts, engineered bacteria carrying the P16090-OLE1 plasmid specifically expressed a protein band corresponding to the expected molecular weight of the OLE1-6xHis-tagged protein. In the control group without bile salts, this band was absent. This strongly demonstrates that our bile salt-responsive promoter P16090 can effectively regulate downstream gene expression. Additionally, the OLE1-DEFB4A co-expression system, fused with a 2A peptide, produced the expected protein products as designed.

3. Function

Building on the confirmation of correct molecular construction, we further conducted quantitative evaluations of the final functionality of the engineered bacteria. For therapeutic functionality, we analyzed the metabolites in the culture supernatant of the engineered bacteria using liquid chromatography. The results showed that engineered bacteria containing the accA gene exhibited a significant increase in short-chain fatty acid production \(e\.g\., acetate and propionate\). Furthermore, engineered bacteria carrying both accA and P16090-OLE1 plasmids displayed a notable increase in oleic acid concentration in the supernatant under bile salt induction. Additionally, in a cell model, the supernatant from bacteria expressing DEFB4A demonstrated a marked ability to mitigate oxidative stress. These findings collectively confirm that the engineered bacteria can synthesize the designed target metabolites and perform their intended physiological functions.

For the biosafety functionality, we designed a time-kill curve experiment to verify the reliability of the Hok/Sok system. When the engineered bacteria were transferred from bile salt-containing medium to bile salt-free medium, the viable cell count dropped sharply to zero within 8 hours, while the control group continued to grow normally. This result strongly demonstrates that our biocontainment system effectively realizes the design logic of "surviving in the gut, self-destructing outside the gut," providing key data support to ensure environmental safety.

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