Source: CORNELL UNIVERSITY submitted to
GENETIC MODIFICATION OF T7 BACTERIOPHAGE FOR THE RAPID, ELECTROCHEMICAL AND BROAD HOST RANGE DETECTION OF E. COLI IN AGRICULTURAL WATER
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1010308
Grant No.
2015-67011-25967
Cumulative Award Amt.
$22,198.92
Proposal No.
2016-05167
Multistate No.
(N/A)
Project Start Date
Mar 1, 2016
Project End Date
Feb 28, 2017
Grant Year
2017
Program Code
[A7101]- AFRI Predoctoral Fellowships
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Food Science
Non Technical Summary
Raw produce has increasingly become a significant source of foodborne outbreaks in the US, accounting for nearly a quarter offoodborne outbreaks, and almost half of outbreak-related illness reported between 1996 and 2010. In several of theseoutbreaks, irrigation or wash water has been identified as the source of contamination. To address this issue, the Food SafetyModernization Act has proposed defining quality standards for agricultural water. The rules will set allowable limits and requirefarmers to regularly monitor generic E. coli as an indicator of water quality. To meet these standards, farmers will need testingmethods that are reliable, rapid, and easy-to-use. We propose genetically engineering bacteriophage T7 to enable rapid,electrochemical detection of E. coli in agricultural water samples. We intended show that: 1) T7 can be genetically modified toallow for the detection of levels of E. coli expected to be found in agricultural water; 2) this detection scheme is comparable tocurrently used water testing methods in terms of sensitivity and time to detection. This project willlay the foundation for the development of rapid, sensitive, and easy-to-use biosensors that can be leveraged to help improve theoverall safety of the farm to table food chain.
Animal Health Component
40%
Research Effort Categories
Basic
20%
Applied
40%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7124010104060%
7124010110030%
7124010200010%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology; 2000 - Chemistry;
Goals / Objectives
1) Successful genetic modification of the bacteriophage T7 as the delivery agent for an enzymatic reporter that can besubsequently expressed in infected E. coli.2) Characterization of reporter enzyme levels and impact of electrochemical signal amplification.3) Correlation of signal to E. coli levels and determination of the assays limit of detection.4) Successful detection by T7-assay of E. coli in agricultural water samples.
Project Methods
Genetic modification of the bacteriophage T7. We are choosing bacteriophage T7 as our delivery agent for various reasons: itsspecificity for E. coli, its rapid infection to lysis cycle, and its history of successful genetic modification and use as a geneticdelivery system. We will leverage commonly used molecular techniques for the genetic modification of T7 to incorporate thereporter enzyme. There are readily available commercially assays to detect the expression of the reporter enzymes we areinterested in incorporating into T7 . We will use theses assays to confirm reporter enzyme overexpression by comparingsamples of: i) unlysed cells, ii) cells lysed by wild type T7; and iii) cells lysed by our modified T7. If these tests suggest eitherpoor reporter enzyme production or significant background noise due to base cellular levels, we will investigate the use ofalternative reporter enzymes. The results of these experiments would be published and presented at a conference.Reporter enzyme signal & correlation to E. coli levels. Based on our laboratory's previous research into traditional T7 phageamplification for bacterial detection, we will incubated various concentrations of a laboratory E. coli strain with varyingconcentration or either the genetically modified T7 or wild type T7. The data from this experiment will allow for the determinationof the earliest time point at which a statistically significant signal is produced. The data will also demonstrate the impact ofstarting phage concentration on detection time and signal strength. The next step for correlating reporter signal to cellconcentration will be to apply the ideal starting phage concentration identified above and do a dilution series of the laboratory E.coli strain. After the incubation time, determined in the previous experiment, we would record the signal from the reporterenzyme. Regression analysis will be used to determine if a significant correlation exists between electrochemical signal of thereporter enzyme and the starting E. coli concentration. This data will also allow us to determine the limit of detection of ourmethod. The final step will be to repeat this second experiment with various E. coli strain to determine if there are significant differences due to strain type. The results of these experiments would be published and presented at a conference.

Progress 03/01/16 to 02/28/17

Outputs
Target Audience:The academic and regulatory community were reached through publications, both peer-reviewed journal and PhD thesis,of the research accomplished throughthe support of this grant. The results of the research were also presented tofood industry and state regulatory via non-public presentations regarding applications of bacteriophage for bacterial detection and therapy. Some of this work was also shared with food manufacturers (New York State Cheese Manufacturers) as I started my position at Cornell to inform them about sepcific areas of bacteriophage application research in my lab. Changes/Problems:The major change was that I transitioned from a Ph.D. student to Assistant Professor. This transition would not have been possible with the support of this grant as it has allowed me to develop into a researcher that was recruited by a topacademic institution. This grant was critical in providing me support asI started up my new lab at Cornell, andI worked to identify andestablishthe capabilities necessary to continue bacteriophage-related research. Because of this support,I am now positioned to not only perform research related to this grant, but to train future Ph.D. and M.Sc. in areas related to NIFA's priorities in the areas of Food Safety and Food Quality. What opportunities for training and professional development has the project provided?This project has played an important role in supportingmytransitionfrom a PhD student at the University of Massachusetts, and developing into an assistant professor within the department of Food Science at Cornell University overthe summer of 2016. I had no summer funding as I started up, so thesupport of this grant allowed meset up my new lab, bring in equipment necessary for meto continue and build on the bacteriophage research supported by this grant, and begin training my new graduate students in the molecular techniques required for bacteriophage modification. How have the results been disseminated to communities of interest?The results of this work have been disseminated via publication in journals and via my PhD thesis. As part my first year at Cornell, I have also been personallysharing the results of this research and its future application with representatives of the food industry and state regulatory officials, as they visit the department or as I go out to meet them. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? As part of my final paper we showed the abilityof bioengineered T7 phage expressing enzymatic reporters to increase the sensitivity of lateralflow assay for the detection of E. coli. .We have demonstrated a greater than 10-fold increase in sensitivity using a phage-based protein reporter, maltose-binding protein, over the detection of replicated T7 phage viron itself, and a greater then 100-fold increase in sensitivity using a phage-based enzymatic reporter, alkaline phosphatase. This increase in sensitivity enabled us to detect 103CFU/mL ofEscherichia coliin broth after 7h, and by adding a filter concentration step, the ability to detect a regulatory relevantE. coliconcentration of 100CFU/100mL in inoculated river water after 9h, where the current standard requires days for results. The combination of the paper fluidic format with phage-based detection provides a platform for the development of novel diagnostics that are sensitive, rapid, and easy to use.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: SD Alcaine, K Law, S Ho, AJ Kinchla, DA Sela, SR Nugen. "Bioengineering bacteriophages to enhance the sensitivity of phage amplification-based paper fluidic detection of bacteria" Biosensors and Bioelectronics 82, 14-19 (2016)
  • Type: Theses/Dissertations Status: Accepted Year Published: 2016 Citation: Alcaine, Samuel D., "Bacteriophage: Bioengineered Bacterial Detection and Applications" (2016). Doctoral Dissertations May 2014 - current. 547. http://scholarworks.umass.edu/dissertations_2/547