Endolysins have emerged as a novel class of antibacterials and are currently under evaluation in clinical trials. Moreover, Czaplewski et al. (2016)1 ranked endolysins based on their clinical impact and technical feasibility as the alternative class of antibacterials with the greatest potential. A unique feature of endolysins is their modular structure and the opportunities to customize endolysins towards their specific application. A major expansion of this modularity principle is the fusion of endolysins to outer membrane permeabilizing peptides, facilitating efficient transfer across the outer membrane of Gram-negative bacteria, followed by rapid cell lysis 2–4.
The scope of this modularity principle is exciting, as it allows engineering of endolysins against any pathogen and customization of the antibacterial properties. However, this progress is empirical and hampered by the tedious cloning procedures. To address this technical barrier, we developed a novel DNA shuffling method, coined the VersaTile Platform. The VersaTile Platform excels in hands-on time, convenience, efficiency and throughput of module shuffling. The technique implies two steps: (1) the construction of a repository of all modules or so-called ‘Tiles’ that one wants to shuffle and (2) the subsequent assembly of a freely chosen selection of these Tiles in a random or predesigned order. Specifically, we have now a repository of 232 Tiles, which enables us to generate over 10 million variants.
Nevertheless, we lacked a simple and cost-effective method to screen this enormous amount of variety. Using automated pipetting robotics, it would cost us more than 2 years and 15 million € to just screen 1 library 5. By combining the VersaTile Platform with a custom developed ultra-high-throughput microfluidics-based screening workflow, we are now able to assess the full potential of this platform. In this presentation, we will give an overview of the different hurdles that had to be taken in the development of the microfluidics workflow.
References
1. Czaplewski, L. et al. Alternatives to antibiotics—a pipeline portfolio review. Lancet Infect. Dis. 16, 239–251 (2016).
2. Briers, Y. et al. Art-175 is a highly efficient antibacterial against multidrug-resistant strains and persisters of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 58, 3774–84 (2014).
3. Briers, Y. et al. Engineered endolysin-based ‘Artilysins’ to combat multidrug-resistant gram-negative pathogens. MBio 5, e01379-14 (2014).
4. Rodriguez-Rubio, L. et al. ‘Artilysation’ of endolysin lambdaSa2lys strongly improves its enzymatic and antibacterial activity against streptococci. Sci. Rep. 6, 35382 (2016).
5. Agresti J. J. et al, Ultrahigh-throughput screening in drop-based microfluidics for directed evolution, PNAS 107, 4004-4009 (2010).