Team:SZPT-CHINA/Future Work

Our goal is to treat the second-degree and third-degree burn wounds. It is expected to reduce antibiotic use and form a barrier that can prevent wound infection by P. aeruginosa. Throughout the project, we realized that much work remained to be done and that experimental data are further needed to support the entire project. In the future, we will still continue to improve the design and construction to achieve our goals. However, we believe that our work is a considerable contribution to the iGEM community. In general, our future work will be focused in four aspects, including 1.the antipseudomonal drug synthesis module, 2.the c-di-GMP signaling and BC film production module, 3. integration, 4. product.
● Antipseudomonal drug synthesis module
At present, we have successfully introduced the drug production module containing SE- and IMM-encoded genes into Gluconacetobacter hansenii ATCC 53582. At the same time, the Pseudomonas-specificity and antibacterial effect of SE protein expressed with different RBS were verified. In addition, to have a persistent resistance of SE to Pseudomonas aeruginosa, we are engineering an anchor protein, which can enhance the attachment of the anti-pseudomonal drug SE to the BC film produced by G. hansenii ATCC 53582.
● C-di-GMP signaling and BC film production module
In the c-di-GMP signaling and BC film production module, our previous expectation is that the film would be produced under near-infrared light, but not under the dark conditions. In our experiments, we firstly verified that the BphS protein can increase the film production by Gluconacetobacter hansenii ATCC 53582 under near-infrared light conditions. Then the bphS was engineered to integrate together with yhjH and fcsR parts that can hydrolyze c-di-GMP, and a few engineered bacteria were screened for better film growth under NIR light exposure than under dark conditions, but we have not yet managed to make engineered bacteria not grow BC films under dark conditions.

To solve this problem, we will make the following two adjustments in the future:
1. Re-screen the parts that can hydrolyze c-di-GMP.
2. Modulate the BphS expression level or the strength of yhjH and fcsR to achieve the purpose that BC film can be produced under near-infrared light irradiation, but not under dark conditions.
● Integration
The goal of the whole project is to engineer a bacterium that can produce BC film under near-infrared light, but not under dark conditions. While treated by the blue light, the engineered bacterium would be lysed with the release of the antibacterial drugs. Then the antibacterial drugs SE are bound to BC film. In this year, we have individually verified the function of three modules, but have not yet integrated all of three modules into Gluconacetobacter hansenii ATCC 53582. So, it remains to verify whether these modules can operate normally if they are combined together. These will be our major work in the future.
● Product
In our product form, the set expectation is to make live bacteria lyophilizer and an oil-in-water system emulsion. We have made great success in the emulsion part, while the survival rate of live bacteria in the lyophilizer is about 20%, which has not reached our expectation. To improve bacterial survival rate in our products, we will continue to search the literature, and identify better agents and conditions to lyophilize G. hansenii ATCC 53582 in the future.