Issue 19, 2016

Genetic optimization of a bacteriophage-delivered alkaline phosphatase reporter to detect Escherichia coli

Abstract

A large fraction of foodborne illnesses are linked to (∼46%) leafy green vegetables contaminated by pathogens harbored in agricultural water. To prevent this, accurate point-of-production detection tools are required to identify and quantify bacterial contaminants in produce before consumers are impacted. In this study, a proof-of-concept model was engineered for a phage-based Escherichia coli detection system. We engineered the coliphage T7 to express alkaline phosphatase (ALP) to serve as the signal for E. coli detection. Wild type phoA (T7ALP) and a dominant-active allele, phoA D153G D330N (T7ALP*) was inserted into the T7 genome, with engineered constructs selected by CRISPR-mediated cleavage of unaltered chromosomes and confirmed by PCR. Engineered phages and E. coli target cells were co-incubated for 16 hours to produce lysates with liberated ALP correlated with input cell concentrations. A colorimetric assay used p-nitrophenyl phosphate (pNPP) to demonstrate significant ALP production by T7ALP and T7ALP* compared to the vector control (T7EV) (p ≤ 0.05). Furthermore, T7ALP* produced 2.5-fold more signal than T7ALP (p ≤ 0.05) at pH 10. Due to the increase in signal for the modified ALP* allele, we assessed T7ALP* sensitivity in a dose-responsive manner. We observed 3-fold higher signal for target cell populations as low as ∼2 × 105 CFU mL−1 (p ≤ 0.05 vs. no-phage control).

Graphical abstract: Genetic optimization of a bacteriophage-delivered alkaline phosphatase reporter to detect Escherichia coli

Supplementary files

Article information

Article type
Paper
Submitted
26 Feb 2016
Accepted
04 Jul 2016
First published
05 Jul 2016

Analyst, 2016,141, 5543-5548

Genetic optimization of a bacteriophage-delivered alkaline phosphatase reporter to detect Escherichia coli

A. A. Jackson, T. C. Hinkley, J. N. Talbert, S. R. Nugen and D. A. Sela, Analyst, 2016, 141, 5543 DOI: 10.1039/C6AN00479B

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