To create a phage that can do this, we need to engineer the phage genome. Unfortunately, we cannot work with X. fastidiosa because of quarantine measures and slow growth . Instead we work with model organisms E. coli and phage Lambda. Phage Lambda is a good model phage as it is well studied and has capsid structures that are similar to those of X. fastidiosa phages, that can be used to fuse proteins to. To edit phage Lambda, we use two novel methods of genome engineering and phage production. Namely yeast based genome engineering and cell free production of phages using a Transcription Translation (TXTL) mixture, which are shown in figure 1. Together, these methods allow genetic engineering and production of phages without use of the host. This makes our work applicable to future work with X. fastidiosa phages.
We were able to assemble genomes in yeast and produce phages using TXTL. While application to engineered phage Lambda was not reached yet, many important steps have been made towards it.
We constructed a 3D model of the untruncated version of the fusion protein with the ribosomal frameshifting sequence (figure 5). In this model, it is observable that the gpD protein can correctly fold and is not sterically disturbed by the fused domain. Moreover, there is a clear spatial separation between the Chitinase A1 and the gpD protein, generated by the presence of the linking peptide. This model, together with previous studies , gives us an indication that Chitinase A1 could be correctly displayed on the phage capsid, giving the phage the ability to bind to Chitin.