Important for the regulation of the gene circuit is the riboswitch controlling the expression of the Acr. Figure 4 demonstrates the control of GFP expression by the riboswitch in two different systems. The pSEVA23 system shows no fluorescence signal when compared to the negative and positive control. To see whether this effect was due to the gene being unfunctional or the riboswitch controlling its expression tightly, mRNA extraction followed by cDNA synthesis and PCR were performed. Figure 5 shows that even though no GFP is visible, GFP transcripts are present. Therefore, we can conclude that even though transcription of GFP takes place, GFP translation is inhibited by the riboswitch. In this way, we demonstrated the robustness of riboswitch mediated translation inhibition, providing a good foundation for the control of the Acr-dCas9 gene circuit.
By constructing the AcrIIA4-dCas9 gene circuit, we could demonstrate two things. First, we were able to repress gene expression significantly by targeting the phage Lambda early transcripts regulatory region (Figure 6). Second, we managed to restore the expression from phage Lambda promoters by induction of Acr expression. The provided library of RBS sequences, characterized for their function in the presented gene circuit, allows for the control of gene expression in different scenarios. Further applications could include phage therapy delivery for other pathogens affecting plants, humans and the biotechnology sector. In this way, we delivered the basis for the controlled phage release, not just for the Xylencer project, but also for future applications involving targeted phage proliferation in chassis organisms and control of gene expression in general.
The attachment to the insects is mediated via chitin-binding proteins, which adhere to the insect stylet. The insect uses the stylet to feed on plants. We demonstrated that PD1764, an adhesion protein derived from X. fastidiosa, shows high binding abilities to chitin as compared to a bovine serum albumin (BSA) control (Figure 8). Since PD1764 is a chitin binding protein from X. fastidiosa it shows the potential of this protein for effective transmission of Xylencer phages.
In a next step, we generated constructs encoding for the fusion of phage Lambda capsid protein gpD to our adhesion protein candidates. As the phage capsid integrity can be negatively influenced by high amounts of capsid fusion protein , we decided to integrate a ribosomal frame shift (RFS) site between the gpd and adhesion protein coding sequence. The RFS should create a mix of WT and fusion protein. By western blot we could confirm the transcription and translation of our fusion proteins in E. coli (Figure 9). The first lane shows the expected fragment size of the gpd and adhesion proteins connected by a flexible linker (CGSGSGSG). In the second lane, single gpD and gpD fusion protein with RFS sites are visible. In this way we demonstrated the successful fusion of our adhesion protein candidates to a phage capsid protein.
With the adhesion assay (figure 8) we could demonstrate the binding ability of our protein to structures found in the insects stylet. Through the western blot we show the fusion of adhesion proteins to phage capsid proteins. Together, these successes represent two major steps towards implementing the Xylencer phage spreading mechanisms via X. fastidiosa insect vectors.