Team:RDFZ-CHINA/Proof Of Concept

Brief Introduction: Because of the limited experiment data, this page solely introduces how our project will be used in reality. The page includes three parts: genetic modification in lab, chain of production, and the system's application in plants. Genetic Modification in Lab As mentioned on the design page, our system can be divided into four parts, including DSF sensor, phage carrier, phage releaser, and virulence reducing system. These four systems will firstly be constructed on pBBR plasmid in an E.coil. Then the engineered plasmid will be placed inside a Xanthomonas using electrotransformation. So far, the Xanthomonas has not come in contact with the Xoo-sp2 phage. Therefore, no genes of the phage are present in the Xanthomonas, which ensures that the engineered Xanthomonas can successfully enter the logarithmic phase without being affected by the DSF pathway we designed. By then, the DSF concentration is extremely high. If the genes of the phage are added earlier, the phage-producing process will be triggered by the elevated DSF concentration and cause massive death among engineered Xanthomonas. Chain of Production This image depicts our plans regarding the production, packaging, transportation, and implementation (reactivation) processes of our product. First, the engineered Xanthomonas will be stored under negative 80 degrees Celsius, in EP tubes containing glycerin. Whenever we receive an order, we add the bacteria in the EP tubes into fermentation equipment, in which the bacteria is allowed to replicate and grow. When the concentration of bacteria in the equipment reaches a certain value, we move on to the next process. We will then add enzymes to the solution containing bacteria to lower the concentration of DSF molecules. FadD1, extracted from Pseudomonas aeruginosa, is a kind of enzyme that could act on long-chain fatty acids. Since DSF molecules are long-chain fatty acids, FadD1 could be one of the enzymes added to decrease DSF concentration. Right after DSF concentration is lowered, phage Xoo-sp2 will be added to the bacterial solution, enabling the phage to inject its DNA into Killer-X. To make transportation and storage easier, we will precipitate the engineered bacteria through refrigerated centrifugation and then dry it, converting Killer-X into powder. The powder will be packed into the vacuum packages and then delivered to our clients. Now, all our clients need to do is dissolve the powder in water, that can then be sprayed into the crops through their irrigation system. The System's Application in Plants When Killer-X is activacted and sprayed onto the rice, the phage genes it carries enter the system. Therefore, whenever harmful Xanthomonus is present and causes the DSF concentration to increase, Killer-X could sense the increasing concentration and release phage.
When DSF molecules are absent, Vc2 riboswitch binds to c-di-GMP, inhibiting the expression of Acr protein. However, dcas12a and sgRNA will continue to be expressed. They will combine to the pathways expressing endolysin and Xanthomonas phages' major tail protein. Consequently, neither endolysin nor major tail protein will be expressed, hence inhibiting the production of phages.
When a large number of DSF molecules are present, the connection between Vc2 riboswitch and c-di-GMP breaks, and the downstream Acr protein is expressed. AcrVA1 is a multiple-turnover inhibitor that triggers cleavage of the target-recognition sequence of the Cas12a-bound guide RNA to irreversibly inactivate the Cas12a complex. Therefore, RNA polymerase can combine with the DNA and express endolysin and major tail protein. The phage grows and replicates within Killer-X rapidly. Killer-X then decomposes, releasing the phages, which enter the external environment and decompose the invasive Xanthomonas. Conclusion In conclusion, if the infectious Xanthomonas tries to enter the logarithmicz period, the DSF molecules it releases will reveal its intentions. Upon detecting infectious Xanthomonas' vicious ambitions, Killer-X will release Xoo-sp2 and kill all the infected bacteria.