Colorimetric detection of Escherichia coli using engineered bacteriophage and an affinity reporter system
Reporter phage systems have emerged as a promising technology for the detection of bacteria in foods and water. However, the sensitivity of these assays is often limited by the concentration of the expressed reporter as well as matrix interferences associated with the sample. In this study, bacteriophage T7 was engineered to overexpress mutated alkaline phosphatase fused to a carbohydrate-binding module (ALP*-CBM) following infection of E. coli to enable colorimetric detection in a model system. Magnetic cellulose particles were employed to separate and concentrate the overexpressed ALP*-CBM in bacterial lysate. Infection of E. coli with the engineered phage resulted in a limit of quantitation of 1.2 × 105 CFU, equating to 1.2 × 103 CFU/mL in 3.5 h when using a colorimetric assay and 100 mL sample volume. When employing an enrichment step, < 101 CFU/mL could be visually detected from a 100 mL sample volume within 8 h. These results suggest that affinity tag modified enzymes coupled with a material support can provide a simple and effective means to improve signal sensitivity of phage-based assays.
KeywordsBacteriophage Fusion E. coli Binding module Cellulose Magnetic
The authors would like to acknowledge Dr. Kevin Nichols for insightful conversations regarding phage-based detection.
The research reported in this publication was supported by the National Science Foundation under Grant No. 1705815 and the National Institute of Biomedical Imaging and Bioengineering (R21EB024623).
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Conflict of interest
The authors declare that they have no conflict of interest.
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Science Foundation or the National Institutes of Health.
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