Issue 17, 2018

Reporter bacteriophage T7NLC utilizes a novel NanoLuc::CBM fusion for the ultrasensitive detection of Escherichia coli in water

Abstract

Rapid detection of bacteria responsible for foodborne diseases is a growing necessity for public health. Reporter bacteriophages (phages) are robust biorecognition elements uniquely suited for the rapid and sensitive detection of bacterial species. The advantages of phages include their host specificity, ability to distinguish viable and non-viable cells, low cost, and ease of genetic engineering. Upon infection with reporter phages, target bacteria express reporter enzymes encoded within the phage genome. In this study, the T7 coliphage was genetically engineered to express the newly developed luceriferase, NanoLuc (NLuc), as an indicator of bacterial contamination. While several genetic approaches were employed to optimize reporter enzyme expression, the novel achievement of this work was the successful fusion of the NanoLuc reporter to a carbohydrate binding module (CBM) with specificity to crystalline cellulose. This novel chimeric reporter (nluc::cbm) bestows the specific and irreversible immobilization of NanoLuc onto a low-cost, widely available crystalline cellulosic substrate. We have shown the possibility of detecting the immobilized fusion protein in a filter plate which resulted from a single CFU of E. coli. We then demonstrated that microcrystalline cellulose can be used to concentrate the fusion reporter from 100 mL water samples allowing a limit of detection of <10 CFU mL−1E. coli in 3 hours. Therefore, we conclude that our phage-based detection assay displays significant aptitude as a proof-of-concept drinking water diagnostic assay for the low-cost, rapid and sensitive detection of E. coli. Additional improvements in the capture efficiency of the phage-based fusion reporter should allow a limit of detection of <10 CFU per 100 mL.

Graphical abstract: Reporter bacteriophage T7NLC utilizes a novel NanoLuc::CBM fusion for the ultrasensitive detection of Escherichia coli in water

Article information

Article type
Paper
Submitted
26 apr 2018
Accepted
11 iyl 2018
First published
02 avq 2018
This article is Open Access
Creative Commons BY-NC license

Analyst, 2018,143, 4074-4082

Reporter bacteriophage T7NLC utilizes a novel NanoLuc::CBM fusion for the ultrasensitive detection of Escherichia coli in water

T. C. Hinkley, S. Garing, S. Singh, A-L. M. Le Ny, K. P. Nichols, J. E. Peters, J. N. Talbert and S. R. Nugen, Analyst, 2018, 143, 4074 DOI: 10.1039/C8AN00781K

This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. You can use material from this article in other publications, without requesting further permission from the RSC, provided that the correct acknowledgement is given and it is not used for commercial purposes.

To request permission to reproduce material from this article in a commercial publication, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party commercial publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements