Reporting on - A novel system to use bacteriophages as tools to detect bacteria

The first project was geared at developing a rapid phage engineering pipeline using cell-free transcription/translation systems. In order to address the limitations of current phage engineering methods that rely on homologous recombination and CRISPR selection, we first developed a whole genome assembly method to create a new engineered bacteriophage variant of K1F phage containing a SpyTag moiety on the capsid head. This method enabled the rapid creation of a number of different engineered K1F variants through an 8 piece Gibson assembly of the genome. Following on from the establishment of the genome assembly method and the creation of the SpyTag K1F phage, we developed methods to allow for the rebooting of the SpyTag K1F phage from its genome in Cell-Free TXTL system. These two methods were then combined to create a rapid phage engineering pipeline, namely the SpyPhage system, in which a single SpyTagged phage can be assembled in a TXTL reaction and subsequently post-translationally decorated with SpyCatcher fused therapeutic proteins. As a proof of concept, we demonstrated for the first time the cell-free assembly and decoration of the phage surface with two alternative fusion proteins, SpyCatcher-mCherry-EGF and SpyCatcher-mCherry-Rck, both of which facilitate the endocytotic uptake of the phages by a urinary bladder epithelial cell line. Overall, our work presents a cell-free phage production pipeline for the generation of multiple phenotypically distinct phages with a single underlying “scaffold” genotype. These phages could become the basis of next-generation phage therapies where the knowledge-based engineering of numerous phage variants would be quickly achievable without the use of live bacteria or the need to repeatedly license novel genetic alterations. 

This work is now published in ACS Synthetic Biology: https://doi.org/10.1021/acssynbio.2c00244

In addition to the above mentioned work, the MLS funds were also utilised for the development of a simple method for the immobilisation of SpyTagged phage on to a PDMS based microfluidic device. During the course of the grant, promising proof of concept work was established that demonstrate the ability of PDMS immobilised phage to capture and infect bacteria on chip. These results would enable the use of the method for the creation of a phage based diagnostic device in the near future. This work is now being compiled into a manuscript and will be submitted to a journal soon.

The funding from MLS proved essential during my PhD and allowed me to meet and exceed my experimental aims, allowing me to publish the work.