caa a22 4500 605498741 CHVBK 20210128100547.0 cr unu---uuuuu 210128e20151001xx s 000 0 eng 10.1007/s00253-015-6867-8 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6867-8 Phage-protease-peptide: a novel trifecta enabling multiplex detection of viable bacterial pathogens [Elektronische Daten] [S.D. Alcaine, L. Tilton, M. Serrano, M. Wang, R.W. Vachet, S.R. Nugen] Bacteriophages represent rapid, readily targeted, and easily produced molecular probes for the detection of bacterial pathogens. Molecular biology techniques have allowed researchers to make significant advances in the bioengineering of bacteriophage to further improve speed and sensitivity of detection. Despite their host specificity, bacteriophages have not been meaningfully leveraged in multiplex detection of bacterial pathogens. We propose a proof-of-principal phage-based scheme to enable multiplex detection. Our scheme involves bioengineering bacteriophage to carry a gene for a specific protease, which is expressed during infection of the target cell. Upon lysis, the protease is released to cleave a reporter peptide, and the signal detected. Here we demonstrate the successful (i) modification of T7 bacteriophage to carry tobacco etch virus (TEV) protease; (ii) expression of TEV protease by Escherichia coli following infection by our modified T7, an average of 2000 units of protease per phage are produced during infection; and (iii) proof-of-principle detection of E. coli in 3h after a primary enrichment via TEV protease activity using a fluorescent peptide and using a designed target peptide for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF MS) analysis. This proof-of-principle can be translated to other phage-protease-peptide combinations to enable multiplex bacterial detection and readily adopted on multiple platforms, like MALDI-TOF MS or fluorescent readers, commonly found in labs. Springer-Verlag Berlin Heidelberg, 2015 Biosensors nationallicence Bacteriophage nationallicence Protease nationallicence Multiplex nationallicence Alcaine S.D. Department of Food Science, University of Massachusetts, 246 Chenoweth Laboratory, 102 Holdsworth Way, 01003, Amherst, MA, USA aut Tilton L. Department of Food Science, University of Massachusetts, 246 Chenoweth Laboratory, 102 Holdsworth Way, 01003, Amherst, MA, USA aut Serrano M. Department of Chemistry, University of Massachusetts, Amherst, MA, USA aut Wang M. Department of Chemistry, University of Massachusetts, Amherst, MA, USA aut Vachet R.W. Department of Chemistry, University of Massachusetts, Amherst, MA, USA aut Nugen S.R. Department of Food Science, University of Massachusetts, 246 Chenoweth Laboratory, 102 Holdsworth Way, 01003, Amherst, MA, USA aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/19(2015-10-01), 8177-8185 0175-7598 99:19<8177 2015 99 253 https://doi.org/10.1007/s00253-015-6867-8 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6867-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Alcaine S.D. Department of Food Science, University of Massachusetts, 246 Chenoweth Laboratory, 102 Holdsworth Way, 01003, Amherst, MA, USA aut NATIONALLICENCE 700 1- Tilton L. Department of Food Science, University of Massachusetts, 246 Chenoweth Laboratory, 102 Holdsworth Way, 01003, Amherst, MA, USA aut NATIONALLICENCE 700 1- Serrano M. Department of Chemistry, University of Massachusetts, Amherst, MA, USA aut NATIONALLICENCE 700 1- Wang M. Department of Chemistry, University of Massachusetts, Amherst, MA, USA aut NATIONALLICENCE 700 1- Vachet R.W. Department of Chemistry, University of Massachusetts, Amherst, MA, USA aut NATIONALLICENCE 700 1- Nugen S.R. Department of Food Science, University of Massachusetts, 246 Chenoweth Laboratory, 102 Holdsworth Way, 01003, Amherst, MA, USA aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/19(2015-10-01), 8177-8185 0175-7598 99:19<8177 2015 99 253