Activity-Fed Translation (AFT) Assay: A New High-Throughput Screening Strategy for Enzymes in Droplets.
There is an increasing demand for the development of sensitive enzymatic assays compatible with droplet-based microfluidics. Here we describe an original strategy, activity-fed translation (AFT), based on the coupling of enzymatic activity to in vitro translation of a fluorescent protein. We show that methionine release upon the hydrolysis of phenylacetylmethionine by penicillin acylase enabled in vitro expression of green fluorescent protein. An autocatalytic setup where both proteins are expressed makes the assay highly sensitive, as fluorescence was detected in droplets containing single PAC genes. Adding a PCR step in the droplets prior to the assay increased the sensitivity further. The strategy is potentially applicable for any activity that can be coupled to the production of an amino acid, and as the microdroplet volume is small the use of costly reagents such as in vitro expression mixtures is not limiting for high-throughput screening projects.
Institut des Sciences de la Vie, Université catholique de Louvain, Place Croix du Sud 4-5, Boîte L7.07.06, 1348 Louvain-la-Neuve (Belgium).|Institut de Science et d'Ingénierie Supramoléculaires (ISIS), Université de Strasbourg, CNRS UMR 7006, 8 Allée Gaspard Monge, 67083 Strasbourg Cedex (France).|École Supérieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI ParisTech), 10 rue Vauquelin, 75231 Paris Cedex 05 (France).
Gabrielle
G
Woronoff
Institut de Science et d'Ingénierie Supramoléculaires (ISIS), Université de Strasbourg, CNRS UMR 7006, 8 Allée Gaspard Monge, 67083 Strasbourg Cedex (France).|Architecture et Réactivité de l'ARN, CNRS UPR 9002, Université de Strasbourg, 15 rue René Descartes, 67084 Strasbourg (France).
Michaël
M
Ryckelynck
Institut des Sciences de la Vie, Université catholique de Louvain, Place Croix du Sud 4-5, Boîte L7.07.06, 1348 Louvain-la-Neuve (Belgium).
Julia
J
Wessel
Institute of Condensed Matter and Nanosciences, Université catholique de Louvain, 1 Place Louis Pasteur, 1348 Louvain-la-Neuve (Belgium).
Olivier
O
Schicke
Institut de Science et d'Ingénierie Supramoléculaires (ISIS), Université de Strasbourg, CNRS UMR 7006, 8 Allée Gaspard Monge, 67083 Strasbourg Cedex (France). andrew.griffiths@espci.fr.|École Supérieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI ParisTech), 10 rue Vauquelin, 75231 Paris Cedex 05 (France). andrew.griffiths@espci.fr.
Andrew D
AD
Griffiths
Institut des Sciences de la Vie, Université catholique de Louvain, Place Croix du Sud 4-5, Boîte L7.07.06, 1348 Louvain-la-Neuve (Belgium). patrice.soumillion@uclouvain.be.
Patrice
P
Soumillion
D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D008958:Models, Molecular; D010399:Penicillin Amidase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D014176:Protein Biosynthesis; D014158:Transcription, Genetic
D049452:Green Fluorescent Proteins; D010399:Penicillin Amidase
droplet-based microfluidics; enzyme catalysis; in vitro translation; penicillin acylase; protein expression