This is one of the most important parts of the project, here is where we present whether or not our project really works as we originally proposed it would. The most awaited moment of the year. After reading all the work done through this project, one question arises: does it really work? In order to give a real and conclusive answer to this specific question, a simulation of close-to-reality conditions was carried out, where leaves were inoculated with two types of Erwinia amylovora, wild and transformed with our composite aiiA BioBrick (BBa_K2471000). Photos were taken through several days of the process in order to visualize the evolution of the disease. We compared the progress of the disease caused by the wild-type E. amylovora with the one caused by the transformed one. The differences were clear and convincing: the disease was dismissed considerably on the leaf inoculated with the transformed E. amylovora. This phenomenon was observed because the aiiA gene encodes for an N-Acyl homoserine lactonase, which degrades N-Acyl homoserine lactones (AHLs), and in turn, disrupts the quorum sensing and virulence factors that come with it. Therefore, we can conclude that our project does indeed work.

Two different types of Erwinia amylovora, wild and transformed with our composite aiiA BioBrick (BBa_K2471000), were inoculated on apple leaves. The disease (Fire Blight) began to be visible three days after inoculation and developed in a gradual and similar manner until the eighth day when the disease considerably increased its expansion through the leaf (Figure 1). However, differences in disease development (both the severity and onset time) were seen between the wild-type E. amylovora (Figure 2) and the one transformed with the aiiA BioBrick (Figure 3). The assay consisted of cutting leaves and inoculating them with the previously mentioned types of E. amylovora -plus a negative control, which was inoculated with medium without inoculum-, in order to simulate close to real conditions and see how each of them evolved.

Erwinia amylovora’s virulence factors principally depend on its cellular density, N-Acyl homoserine lactones (AHLs) are responsible for and involved in the synthesis of many secondary metabolites, among them, exopolysaccharides like the amylovoran and levan. AHLs in E. amylovora has been proven to contribute to the expression of several virulence factors and disease development; similar to the role they play in other pathogenic bacteria. When incorporating the aiiA BioBrick (BBa_K2471000) into E. amylovora, leaf necrosis was inhibited (Figure 3), when compared with the wild-type E. amylovora (Figure 2). The production of the characteristic bacterial exudate of the disease was inhibited, which is composed of the amylovoran and levan EPS, and the infection that is presented by the type III secretion system dismissed.

The gene expressed is an N-Acyl homoserine lactonase, which is involved in the hydrolysis of AHLs. The degradation of these molecules stops the AHL-EamR complex from binding, which in turn, stops the activation of an operon responsible for the EamI transcription of intracellular AHLs and other genes that contribute to several virulence factors.

Phenotypic dismissal of Fire Blight was observed when the disease was caused by E. amylovora electroporated with our aiiA BioBrick compared to the wild-type E. amylovora. These results show a noticeable decrease in disease development in the bacteria transformed with our BioBrick (Figure 3). Thus, we can conclude that our gene does reduce the decay caused by the disease in close-to-reality scenarios.