Source: CORNELL UNIVERSITY submitted to
ENGINEERING BACTERIOPHAGE FOR THE ULTRA SENSITIVE DETECTION OF FOOD BORNE AND ANIMAL PATHOGENS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1010727
Grant No.
2016-67011-25969
Cumulative Award Amt.
$79,000.00
Proposal No.
2016-09640
Multistate No.
(N/A)
Project Start Date
Apr 1, 2016
Project End Date
Dec 31, 2018
Grant Year
2017
Program Code
[A7101]- AFRI Predoctoral Fellowships
Project Director
Hinkley, T.
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Food Science
Non Technical Summary
Thetop 14 foodborne pathogens in the United States create almost 9million infections per year and almost half (~46%) are linked to leafygreen produced contaminated from soiled agricultural water. Advancements in rapid, sensitive and affordable point of care diagnostics serve to greatly impact the public health system. I developinggenetically engineeredbacteriophages to be used in bacteriophage-based diagnostic assays that sensitively detect foodborne and animal pathogens. My overall goal is to improve public health & public safety through the sensitive detection of harmful pathogens present in foods and drinking water. As a result, the potential damage, cost, and time lost associated with foodborne outbreaks will all be decreased.
Animal Health Component
50%
Research Effort Categories
Basic
25%
Applied
50%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71240301080100%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4030 - Viruses;

Field Of Science
1080 - Genetics;
Goals / Objectives
My overall goal is to genetically engineer specific viruses that only infect bacteria (bacteriophages) to serve asbacterial recognition systems for improved food safety and animal health.Objectives:1. Insert reporter enzymes into bacteriophage genome2. Develop a CRISPR based selection mechanism to isolate mutant bacteriophages.3. Increase expression levels of reporter enzymes to decrease expression bacterial detection limits.4. Evaluate the effectiveness of other repoter enzymes.5. Evaluate the effectiveness of creating reporter enzyme fusion proteins.
Project Methods
Efforts: Formal classroom instruction (TA & guest lectures)Laboratory instruction of graduate student lab members and undergraduate mentees. Development of streamlined method using CRISPR for genetically engineering wild type bacteriophages.Extension & outreach to local farmersat the end of the project once the recombinant phage has been introduced into a fully developed device.Evaluation: Each of the genetically engineered phages will be evaluated for limit of detection as compared to other phages in our lab.Enzyme activity and concentration will be evaluated by classic enzyme kinetic studies to determine the best candidate reporter enzyme.

Progress 04/01/16 to 12/31/18

Outputs
Target Audience:The target audience for this research is graduate students, professors, and professionals in the field. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Conference Talks Posters available in Cornell hallways What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Reporter genes were inserted into bacteriophage genomes 2. Selection mechanisms were used to isolate recombinant bacteriophages. 3. Various regulatory sequences were assayed to determine the optimal reporter enzyme expression. 4. Multiple reporter enzymes were assayed 5. An effective reporter enzyme fusion decreased detection limits.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Hinkley, T. C., Singh, S., Garing, S., Le Ny, A. L. M., Nichols, K. P., Peters, J. E., ... & Nugen, S. R. (2018). A phage-based assay for the rapid, quantitative, and single CFU visualization of E. coli (ECOR# 13) in drinking water. Scientific reports, 8(1), 14630.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Hinkley, T. C., Garing, S., Singh, S., Le Ny, A. M., Nichols, K. P., Peters, J. E., ... & Nugen, S. R. (2018). Reporter bacteriophage T7 NLC utilizes a novel NanoLuc:: CBM fusion for the ultrasensitive detection of Escherichia coli in water. Analyst, 143(17), 4074-4082.


Progress 04/01/16 to 03/31/17

Outputs
Target Audience:Individuals - Students/Faculty/Staffin the Cornell Food Science department, including undergrads, graduate students, postdoctoral researchers as well as laboratory technicians/lab managers. Groups - Published research displayed on Cornell's walls for general audience viewing. Presentation of material at conferences, such as the American Chemical Society (ACS) among others. Changes/Problems:The biggest change to the project is to prioritize screening methods over selection after multiple CRISPR selections proved to be non-effective. Additionally, some bacteriophages have developed natural CRISPR defense mechanisms. Experiments are planned to optimize the CRISPR selection system but will always be coupled with screening methods to ensure the proper recombinants are selected. What opportunities for training and professional development has the project provided?As this project involves working with genetics in addition to culturing bacteria and bacteriophages, I have had the opportunity to train our new lab members in many of these techniques. The transfer to Cornell (setting up a new lab) has allowed me to aid in the writing of standard operating procedures in addition to consultation on new equipment purchases. How have the results been disseminated to communities of interest?I will present current research findings at the 253rd ACS in San Francisco held in the first week of April 2017. What do you plan to do during the next reporting period to accomplish the goals?1. Insert more novel reporter enzymes into bacteriophage. 2. Optimize a positive selection strategy such as CRISPRto improve the efficiency of recombination. 3. Continue biopanning to increase expression levels. 4 & 5. Continue to evaluate novel reporter enzyme fusions and optimize their effectiveness in final assay conditions.

Impacts
What was accomplished under these goals? 1. Success - Multiple reporter enzymes have been inserted into the bacteriophage genome. 2. Experiments with CRISPR based selection have been inefficient and add little benefit as compared to its added complexity. Colorimetric screening methods have proven to be the most useful in the identification of recombinant bacteriophages. 3. Multiple rounds of biopanning were performed using a colorimetric screening method to isolate the bacteriophages that produced the strongest signal. 4. Experiments have been completed with acid phosphatase, alkaline phosphatase, and luciferase. Partial optimization of reaction conditions and substrates have been achieved. 5. The addition of an affinity tag (carbohydrate binding module, his-tag) has shown to aid in the concentration of signal.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Jackson, A. A., T. C. Hinkley, J. N. Talbert, S. R. Nugen and D. A. Sela (2016). "Genetic optimization of a bacteriophage-delivered alkaline phosphatase reporter to detect Escherichia coli." Analyst.