Engineering Cells for Optimised Phage Therapy 1=Engineering 2=Biomaterials and Tissue Engineering

The key engineering contribution of this project will be in the development of a transcriptional-translational (TXTL) platform and in its optimisation. Cell-free transcription-translation is becoming an effective technology for in vitro synthetic biology and bioengineering. In recent years, DNA-dependent in vitro protein synthesis has rapidly expanded its range of applications to research areas such as biological network prototyping by accelerating the build-design-test cycle, artificial cell systems and biological physics, nanotechnologies, metabolic and chemical engineering, medicine, and the production of functional membrane proteins. In vitro protein synthesis is increasingly employed as a means to construct, understand and interrogate complex biochemical systems, from molecular to cell-sized scales. The hybrid bacteriophage-Escherichia coli system, invented in the 90s, is still the most popular cell-free TX-TL platform. Commercially available, this system is useful as an alternative to recombinant protein expression and for molecular applications such as protein evolution, proteomics, and production of therapeutics. Transcription is performed by a bacteriophage RNA polymerase with its promoter, usually T7, due to its simplicity, high specificity, and strength. The translation machinery is provided by a cytoplasmic extract, often from E. coli. In those systems, protein synthesis can reach more than 1 mg/mL The PURE system, also based on T7, allows working in a simpler environment than extract-based systems. While hybrid T7 systems are useful for a vast array of applications, their transcription consists of only a few elements-a serious limitation in a thriving era of synthetic biology and gene circuit engineering. Elementary gene circuits have been executed in bacteriophage cell-free systems, but those platforms are not well suited for the construction of complex in vitro dynamical systems programmed with DNA as working with so few promoters is too limiting. The development of a synthetic T7 transcription toolbox could provide an alternative to this limitation, although it has to be tested in cell-free conditions. In that perspective, the development of an all E. coli cell-free TX-TL system that recapitulates the entire sigma factor transcription scheme was a major improvement to the existing in vitro protein synthesis technology. By expanding transcription to hundreds of regulatory elements, from E. coli and other bacteria, this platform has proven useful for numerous applications, especially for testing synthetic and natural genetic parts, for prototyping gene circuits and for constructing minimal cell systems.

In this project, the student will build and characterise a unique cell-free TXTL portal for the construction of complex biochemical systems through the execution of DNA programs in vitro. The target application would be to create a pathogen-specific fluorescent phage that can be used in a clinical environment to detect the specific bacteria a patient has been infected with.