Notebook

As members of the GEC-Guangzhou team, we have conducted a series of experiments focusing on three major systems: a sensing system, a therapeutic system, and a suicide system. These systems were developed and tested using engineered E. coli strains, with each system designed to address specific functional goals within our project.

The sensing system aimed to develop a mechanism in E. coli capable of detecting Initially, we designed a sensing system targeting tetrathionate, incorporating a plasmid with inducible promoters to express mRFP (red fluorescent protein) as the reporter gene. After conducting experiments with varying tetrathionate concentrations, we found that the response was insufficient and inconsistent, leading to a decision to shift our focus toward developing a thiosulfate sensor. In the new system targeting thiosulfate, we constructed a more sensitive plasmid incorporating improved promoters and downstream linkage to LacZ. LacZ enzymatically breaks down X-gal, producing a visible blue product that allowed for easier detection and measurement of the sensor’s performance. Extensive tests were conducted with different concentrations of sodium thiosulfate demonstrating a robust, dose-dependent increase in fluorescence and colorimetric output.

Our therapeutic system focused on constructing plasmids capable of expressing therapeutic proteins, such as hEGF (human epidermal growth factor), in E. coli. The goal was to optimize the expression and secretion of hEGF using engineered secretion pathways. Various plasmid constructs were created, including those with inducible promoters controlling the expression of hEGF. The experiments included testing the protein's secretion levels in the bacterial cultures and assessing the biological activity of hEGF on mammalian cell lines. ELISA assays confirmed the presence of secreted hEGF, and subsequent activity tests showed significant positive effects on cell growth and viability, validating the system’s potential as a therapeutic tool.

The suicide system was designed as a safety mechanism to control bacterial growth. It employed the mazE-mazF toxin-antitoxin system, where the toxin gene mazF was activated by an arabinose-inducible promoter, while the antitoxin mazE was controlled by a rhamnose-inducible promoter. Upon activation, mazF expression led to cell death unless counterbalanced by mazE expression. The system was rigorously tested through growth inhibition assays, which measured OD600 to track bacterial survival under various induction conditions. The results confirmed that the system functioned as intended, with induced expression of mazF leading to significant bacterial cell death, providing an effective biocontainment strategy.

These three systems—sensing, therapeutic, and suicide—form the core of our project, each playing a critical role in demonstrating the versatility and safety of engineered bacterial systems for potential applications in environmental sensing and therapeutic delivery.