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Team:HKUST-Hong Kong/Design Overview
From 2012.igem.org
Design Overview
In this project, we would to introduce our genetically engineered Bacillus subtilis, B. hercules, as an anti-colon-tumor agent to provide direct tumoricidal effect during cancer therapy. It is applied as oral medicine which retains viability through digestive tract and executes anti-tumor activity when and only when it is binding to colon cancer cell. Three modules are designed and assembly together to achieve our final goal.
1. Target Binding
Driven by Pveg constitutive promoter, RPMrel, the colon-tumor specific peptide is designed to be displayed on the cell wall of B. subtilis under the facilitation of LytC cell wall displaying system. With this peptide displaying, B. subtilis is taken orally by patient. It is expected to reach colon without retaining or colonizing in gastrointestinal tract. However, once it reaches colon, it will be held up around colon tumor cells and colonize around them, waiting for signal to produce anti-tumor molecule 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 colon cancer cell. 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 under xylose inducible promoter originated from Bacillus megaterium. Since no xylose is present in colon, xylose coated in enteric capsules can be taken orally to induce the production of BMP2 in colon when B. subtilis 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 controlling the timing and dosage of anti-tumor cytokine produced in colon. Xylose inducible promoter is used in our project to determine the time of BMP2 produced. When and only when B. subtilis has bound to colon tumor cell, will BMP2 production be initiated.
While the locally concentrated BMP2 is expected to suppress colon tumor grow and trigger the apoptosis of tumor, the adverse effect from over-dose BMP2 is also designed to be prevented from a toxin-antitoxin system. While antitoxin YdcD is produced under the control of a low efficiency promoter Ptms, YdcE (EndoA, toxin) is designed to be expressed simultaneously with BMP2 under the driving of xylose inducible promoter. When BMP2 is intensively produced under the inducing from xylose, the expression of YdcE (EndoA, toxin) will overwhelm the protection threshold from antitoxin YdcD (EndoAI). The overexpression of toxin with BMP2 production will therefore cause the growth inhibition of B. subtilis and stop any protein synthesis under this stress condition. For detailed description, please refer to module: Regulation and Controlling System.
