Team:HKUST-Hong Kong/Design Overview

In this project, we would like to introduce our genetically engineered Bacillus subtilis, B. hercules, as an anti- tumor agent to suppress colon tumor growth during cancer therapy. It is to be applied as an oral medicine which retains viability through the digestive tract and executes anti-tumor activity when - and only when - it is binding to colon cancer cells. Three modules are designed and assembled to achieve our final goal.

1. Target Binding

Driven by Pveg constitutive promoter, RPMrel, the colon-tumor binding peptide is designed to be displayed on the cell wall of B. subtilis under the facilitation of LytC cell wall display system. With this peptide displayed, B. subtilis is taken orally by patient. The engineered bacterial cells are expected to reach colon without colonizing the upper gastrointestinal tract. However, once it reaches the colon, it will be held up around colon tumor cells and colonize, waiting for the signal to produce anti-tumor molecule and secrete it to the local environment. For detailed description, please refer to module: Target Binding Module

2. Anti-tumor molecule Secretion:

BMP2 (Bone morphogenetic protein 2) has been reported to be a colon tumor suppressor. It arrests cells in G1 phase and triggers the apoptosis of cancerous colon epithelial cells. To enable the production and secretion of BMP2, type I signaling peptide from secretion protein in B. subtilis is fused to the N- terminus of mature BMP2 originated from mouse genome. The expression of this fusion protein is controlled under xylose inducible promoter originating from Bacillus megaterium. Since no xylose is normally present in colon, xylose coated in enteric capsules can be taken orally to induce the production of BMP2 in colon when the B. subtilis vector has successfully localized around colon tumor. For detailed description, please refer to module: Anti-tumor Molecule Secretion

3. Regulation and control System:

Two regulatory systems are designed in order to control the timing and dosage of anti- tumor cytokine production in the colon. Xylose inducible promoter is used in our project to determine the timing of BMP2 production. Only when the genetically modified B. subtilis has bound to colon tumor cells will BMP2 production be initiated.

While the locally concentrated BMP2 is expected to suppress colon tumor growth and induce tumor cells to enter apoptosis, the possible adverse effects from BMP2 overdose should also be taken into consideration. In our design, this is made preventable by introducing a toxin-antitoxin system. When the antitoxin YdcD is produced under the control of a low efficiency promoter Ptms, YdcE (EndoA, toxin) is designed to be expressed simultaneously with BMP2, driven by the xylose inducible promoter. When BMP2 is intensively produced under xylose induction, the expression of YdcE (EndoA, toxin) will overwhelm the protection limit of antitoxin YdcD (EndoAI). The overexpression of toxin together with BMP2 production will therefore inhibit the growth of B. subtilis, and result in the termination of protein synthesis activity. For detailed description, please refer to module: Regulation and Controlling System.