Team:Richmond UR/Design
Design
Based on our previous published work, including Bordeaux 2015’s work, we know that curdlan can be induced for production in wild-type Agro. Therefore, we know the genes required to produce it. However, curdlan is only produced in certain instances. In order for our strain to induce immunity in plants, the curdlan genes need to be upregulated at a precise time, ideally when the bacteria comes into contact with a plant.
How can we engineer Agro to produce curdlan precisely when we want to increase plant immune defenses? Using AgroImmunity!
Our plan is to create a non-pathogenic strain of Agrobacterium that contains only the plant-wound-recognition parts of the virulence pathway. In our engineered AgroImmunity, these genes will induce curdlan production when Agro encounters a wound site. In this way, AgroImmunity can act to induce the wounded plant to defend itself from any pathogenic Agrobacterium that are in the rhizosphere.
The plan for creating AgroImmunity is pictured above. You’ll notice the use of a promoter, PN25, rather than their endogenous promoters. Previous published work in Agrobacgterium has shown that PN25 is a more ideal promoter. Successfully creating this part in a BioBrick was an additionalgoal of the summer.
Once we had the required biobricks, our goal was to make a shuttle vector to then transmit our gene of interest into the Agro genome. We are using the idea of homologous recombination to do this. Although the concept is most commonly related to cell division, it can also prove useful in other instances. In our case, we would use it to insert our Biobrick into the Agro genome, switching it with the normal gene for curdlan production. That way, once the Vir genes would be triggered, curdlan would be produced. Images of our plan are below.
We have designed a shuttle vector that will help us to create AgroImmunity. This shuttle vector backbone (pKmobsacB) carries the gene for Kanamycin resistance and contains the sacB gene, which allows for negative selection of the plasmid after homologous recombination with the genome.
The shuttle vector was designed using TWIST and contains homology to the Agro genome on either side of the curdlan promoter. In between these areas of homology we have synthesized several genes, including Spectinomycin resistance, VirAY293F and VirG under the control of the PN25 promoter, and lacZ under the control of the vir promoter.
Two rounds of homologous recombination can take place on either side of the endogenous curdlan promoter.
Once homologous recombination has taken place, the engineered AgroImmunity strain should be Spectinomycin resistant. By growing these candidate colonies in the presence of sucrose, we can negatively select against cells that still contain the shuttle vector with the sacB gene.
The final Agroimmunity creation will respond to plant signals through VirAY293F, which is more sensitive to phenols, and sugar-bound ChvE. This will initiate phosphorylation of the transcription factor VirG, which will induce the production of Beta-galactosidase through the lacZ gene and, most importantly, curdlan production. The produced curdlan will cause the plant to produce an immunological response that will prevent pathogenesis from virulent Agrobacterium in the rhizosphere.