A multiplexed cell-free assay to screen for antimicrobial peptides in double emulsion droplets

Abstract

The global surge in bacterial resistance against traditional antibiotics triggered intensive research for novel compounds, with antimicrobial peptides (AMPs) identified as a promising candidate. Automated methods to systematically generate and screen AMPs according to their membrane preference, however, are still lacking. We introduce a novel microfluidic system for the simultaneous cell-free production and screening of AMPs for their membrane specificity. On our device, AMPs are cell-free produced within water-in-oil-in-water double emulsion droplets, generated at high frequency. Within each droplet, the peptides can interact with different classes of co-encapsulated liposomes, generating a membrane-specific fluorescent signal. The double emulsions can be incubated and observed in a hydrodynamic trapping array or analysed via flow cytometry. Our approach provides a valuable tool for the discovery and development of membrane-active antimicrobials.

A comprehensive review of Microfluidic approaches in cell-free synthetic biology

“…Microfluidic devices have proven to be absolutely essential in many of these developments and a great variety of research over the last decade focused on utilizing microfluidics to tackle challenges in cell-free systems. There is a growing body of research on compartmentalization of cell-free reactions in microdroplets (Petra et al, 2005;Holstein et al, 2021) and hydrogels (Park et al, 2009;Cui et al, 2020), construction of artificial vesicles to imitate cellular membranes (Arriaga et al, 2014;Deshpande et al, 2016), co-encapsulation of liposomes (Stucki et al, 2021a;Nuti et al, 2022) and coacervates (Pir Cakmak et al, 2019;Abbas et al, 2022) to imitate intracellular organelles and establishing cellular processes such as cell growth (Scott et al, 2016;Bhattacharya et al, 2019), division (Deshpande et al, 2018;Steinkühler et al, 2020;De Franceschi et al, 2024), fusion (Gong et al, 2008;Caschera et al, 2011) and transcription-translation (TX-TL) (Vincent and Libchaber, 2004;Vincent et al, 2005) within these artificial cell constructs. There have also been applications of microfluidic devices for high-throughput characterization of binding affinities (Maerkl and Quake, 2007;Swank et al, 2019) and microfluidic chemostats enabling continuous steady-state cell-free reactions (Niederholtmeyer et al, 2013;Karzbrun et al, 2014;Lavickova et al, 2020;Laohakunakorn et al, 2021;Swank and Maerkl, 2021).…”

“…These advantages have seen implementation of double emulsions as compartments in several cell-free studies. For example, Nuti et al encapsulated artificial vesicles in DEs to screen for activity of anti-microbial peptides (AMPs) (Nuti et al, 2022). Since AMPs are known to disrupt bacterial membranes and also show slight toxicity towards mammalian cell membranes (Magana et al, 2020), CFPS is a good alternative for their production and testing.…”