PHACTORY

Abstract

Antimicrobial resistance is a major emerging threat as reported by the WHO. Worldwide implementation of bacteriophage therapy, a 100-year old treatment method employing the natural enemies of bacteria, is impeded by the lack of common manufacturing procedures to meet international quality and safety standards. Based on synthetic biology we created PHACTORY, a cell-free molecular assembly line for bacteriophages. We demonstrate expression of several phages including T7, MS2 and 3S at clinically relevant concentrations. Exploiting the open nature of cell-free systems, PHACTORY enables modular composition of bacteriophages with engineered proteins while remaining GMO-free. We developed a quality control apparatus and utilize state-of-the-art bioinformatics, as well as purification and encapsulation protocols. To expand our production variety/compatibility while reducing cost, we optimized and engineered home-made E. Coli cell extract. Compared to traditional manufacturing procedures, PHACTORY yields ready-to-use bacteriophages in a fraction of the production volume while requiring S0 instead of S2+ biosafety levels.

Cell-free extract engineering

One Problem of cell extract, is the stability of linear DNA. In escherichia coli, endogenous and exogenous linear DNA are differentiated by specific proteins, such as the recombinase complex RecBCD. It recognizes genomic double strand breaks via a specific sequence of 8 bases called Chi-site and prepares the DNA for homologous repair. However, foreign DNA lacks the Chi-site and is therefore degraded by RecBCD. For experiments in cell extract expensive and inefficient inhibitors are used. Since a knockout of RecBCD is lethal, we chose to modify the enzyme to enable selective depletion after cell lysis. Therefore, we came up with several depletion strategies:

Cell free protocol optimization

The financial advantage of producing home-made cell extract for in vitro protein expression is virtually reversed by the fact that it is tedious and time-consuming, with huge batch to batch variations of the resulting product. Therefore, our objective is to optimize the protocol for self-made cell extract from E. coli, by simplifying time consuming steps while eliminating the central sources of variation during the preparation process. The focus of our effort is to determine the influence of cultivation conditions on extract quality and optimizing the cell lysis for speed, protein yield and nal product quality. By implementing novel ways of extract characterization, we aim to gain a deeper understanding of cell-free systems.

Phage engineering and phage assembly in TX/TL

Generation of a universal platform for phage engineering: Our TX-TL system is superior to conventional bacterial based phage production in the following aspects:
 - Assembly of phages in vitro
 - Exchange of phage fibers in vitro
 - Establishment of homogeneous phage batches
 - Reduction of endotoxins within phage batches We will exemplarily engineer a non lytic fluorescent T7 phage, which targets a non primary E. coli strain.

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