Team:RDFZ-CHINA/Implementation
1. Proposed End Users and Commercialization
Our product Killer-X is an engineered bacterium that can inhibit the proliferation of Xanthomonas, and will be sprayed onto the plants using drones. Our proposed end-users of Killer-X are the farmers, professionals, and pesticide companies who treat Xanthomonas infected plants. The final powder product is convenient to operate for farmers, just like traditional chemical pesticides. However, they still need instructions from pesticide companies, so they are also engaged in our commercialization. For pesticide companies, our image is to cooperate and further improve our products, to transform monomer injection into large-area and high-efficiency applications.
2. Engineering Implementation of Our Project
a. Killer-X can effectively inhibit the progagation of Xanthomonas
According to our design page(https://2021.igem.org/Team:RDFZ-CHINA/Implementation), we can see that the experimental group which added Killer-X has a lower bacteria colony density and the disease symptoms of the experimental group were much alleviated than the control group. This proves that our program can be used to treat Xanthomonas infections, and we can work with drug companies to further develop our programs to solve this knotty plant disease problem.
b. Proper time for phage carrying system to release the phage
We have negative feedback during our design of pathways. The Phage carrying system is a pathway to control when the phage should be released. In our pathway, the phages will grow rapidly in our engineered Xanthomonas when the DSF sensor has a high concentration. The engineered Xanthomonas will be broken down, and the phages enter the environment to clean up the invading Xanthomonas. This negative feedback mechanism makes the whole system more rational, releasing the corresponding number of phages according to the actual situation.
3. Safety
In view of the fact that the engineering bacteria we chose – the Xanthomonas - are toxic to plants. This may in turn affect other plants which are normal during the use of the Killer-X. So, we tried to attenuate the toxicity of our engineering Xanthomonas. Through reading the document, we found a self-stopping mechanism in Xanthomonas: GGDEF-domain protein (GdpX1). We discovered that if GdpX1 protein was overexpressed in Xanthomonas, the toxicity of Xanthomonas decreased significantly. GdpX1 protein basically conforms to our assumption of low toxic Xanthomonas, so we transfer the GdpX1 gene sequence overexpression pathway in engineering Xanthomonas to decrease the toxicity of engineering Xanthomonas.
4. Challenges
a. Our engineering bacteria, Xanthomonas Campestris (XCC) and Xanthomonas Oryzae (XOO), don't have resistance. They may be infected by other bacteria during the culture of our engineering bacteria and thus can lead to experimental failure. During the experiment process, miscellaneous bacteria may be interfered, which would bring upon inaccuracies to our results.
b. We need to transfer plasmids from XCC and XOO by electric transformation. This particular method has a higher probability of failure compared to E. coli.
c. The culture medium of XCC and XOO was different from the E. coli NA medium so that we need to shorten the culture period of Xanthomonas. This makes our experiment more compact.
d. Xanthomonas may have an effect on our fluorescent reporter genes in terms of manipulation and signal detection.
a. Our engineering bacteria, Xanthomonas Campestris (XCC) and Xanthomonas Oryzae (XOO), don't have resistance. They may be infected by other bacteria during the culture of our engineering bacteria and thus can lead to experimental failure. During the experiment process, miscellaneous bacteria may be interfered, which would bring upon inaccuracies to our results.
b. We need to transfer plasmids from XCC and XOO by electric transformation. This particular method has a higher probability of failure compared to E. coli.
c. The culture medium of XCC and XOO was different from the E. coli NA medium so that we need to shorten the culture period of Xanthomonas. This makes our experiment more compact.
d. Xanthomonas may have an effect on our fluorescent reporter genes in terms of manipulation and signal detection.
