Requirement Analysis:

Selecting the appropriate promoter to control the expression of anti-tumor proteins.

Design:

Three distinct promoters capable of activation in the tumor microenvironment – lldR (for high lactate environment, BBa), pPtet (for low oxygen environment, BBa), and Pcadc (for low pH environment, BBa) – were selected for individual experiments designed for each promoter. The design involved detecting promoter activation efficiency by measuring the luminescence intensity of luciferase expressed by the promoter. Subsequently, the aforementioned three promoters were employed to express the anti-tumor protein CDD-iRGD (BBa) with a 6*His tag, to assess their capability to express the target anti-tumor protein.

Development:

Synthesize and clone the promoters into plasmids, then insert them into the engineered bacteria, enabling them to express either luciferase or the anti-tumor protein CDD-iRGD.

Testing:

By measuring the luminescence intensity of bacteria expressing luciferase (Figure 1) and conducting Western blot experiments for the expression of CDD-iRGD protein (Figure 2), the optimal expression environment and expression capability of each promoter were confirmed.

Feedback and Improvement:

Based on the test results, the decision was made to proceed with the aforementioned three promoters.

Requirement Analysis:

Based on the results of the first cycle, we have selected the most suitable promoters and determined the expression requirements for the anti-tumor protein.

Design:

Through discussions with professors, we confirmed the use of a 3-input (BBa) AND gate circuit from two potential options. We also devised an expression plan for the anti-tumor protein, which includes selecting the appropriate engineered bacterial strain DH5α and its cultivation conditions.

Development:

Following the design plan, we constructed the engineered bacteria and inserted the selected promoters and AND gate circuits into them. We ensured that the coding sequence for the anti-tumor protein was correctly embedded in the genome of the engineered bacteria.

Testing:

Cultivated the engineered bacteria with the 3-input AND gate circuit and observed the expression of the anti-tumor protein. Conducted anti-tumor activity tests to verify its efficacy. Additionally, it was co-cultured with 3D tumor spheroids to assess its anti-tumor function.

Deployment/Manufacturing:

Establish stable bacterial strains for repeated experiments.

Maintenance and Support:

Continuously monitor the production process of the engineered bacteria, providing maintenance and support as needed to ensure a consistent supply of the anti-tumor protein.

Feedback and Improvement:

Based on experimental and production outcomes, continuously refine the performance and expression efficiency of the engineered bacteria.

These two engineering cycles worked in tandem, aiding us in the step-by-step development of engineered bacteria targeting tumors, while optimizing the expression of the anti-tumor protein. This iterative approach facilitates ongoing improvements in design and production processes, aligning with the project's needs and objectives.