Engineering bacteriophage for the ultra sensitive detection of food borne and ani

Project Director
Hinkley, T.

Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003

Performing Department
Food Science

Non Technical Summary
Foodborne pathogens create enormous societal costs as a result of their ability to cause significant human suffering and loss of life. The top 14 foodborne pathogens in the United States create almost 9 million infections per year.Detection of these harmful microorganisms is paramount to our strength as a society. Our long-term goal is the improvement of food safety and animal health through improved pathogen detection systems.Our approach for improving pathogen detection systems will involve utilizing newly developedmolecular biological techniques to genetically modify a virus that only infects bacteria (bacteriophage). This genetic modification will force target pathogens (upon phage infection) to express a unique reporter enzyme as a positive indicator for the presence of pathogens. Optimizing reporter enzyme expression levelswill allow for lower bacterial detection limits to be reached. Phages incapable of replication will be constructed to preserve the integrity of the cell and to minimize the potential of environmental contamination.A major goal of this project is to reproduciblydetect pathogenic foodborne bacteria in concentrations less than the required dose for illness. The ultimate goal is to design a detection scheme capable of single organism detection that will provide compliance with many zero-tolerance regulations. This project will dramaticallyimprove the capabilities of low cost diagnostic platforms.

Animal Health Component

30%

Research Effort Categories

Basic

50%

Applied

30%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4010	1040	100%

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;

Keywords

bacteriophage t7

biosensor technologies

genetic engineering

pathogen detection

Goals / Objectives
The long-term goal is to sensitively detect very low concentrations of pathogenic foodborne bacteria that negatively impact public health.The short-term goal is utilize genetic engineering techniques to increase expression of alkaline phosphatase to facilitate single organism detection. 1. Develop a molecular biologicalstrategy using CRISPR/Casto genetically modify bacteriophage T7.2. Develop a strategy using homologous recombinationto genetically modify bacteriophage T7.3. Develop a strategy using restriction enzyme digeststo genetically modify bacteriophage T7.4. Utilize CRISPR/Cas and/or colorimetric substrate technology for selection of desired recombinants. 5. Optimize reporter gene expression by varying insert placement in T7 phage genome.6. Optimize reporter gene expression by introducing genetic insertconcatamers to produce multiple copies at once.7. Purify and characterize reporter enzymes.8. Remove phage replication abilities.

Project Methods
1. Novel phage genetic engineering procedures will be evaluated through PCR confirmation of genetic insertion and full DNA sequencing.2. The concentration of reporter enzyme expression per bacterial cell will be evaluated through standard Michaelis-Menten enzyme kinetics.3. Decreases in detection limits will be evaluated by traditional plating techniques to elucide bacterial populations and how the enzyme kinetic evaluations respond to changes in bacterial populations.4. A non-replicative T7 phage will be designed and evaluated through affinity purifications, full genome sequencing and PCR insert confirmation.5. A special bacterial cell host will be designed as the only location for non-replicative phage production and will be evaluated through full genome sequencing and PCR insert confirmation.

Progress 04/01/16 to 03/31/18

Outputs
Target Audience: Nothing Reported Changes/Problems:No work was done on this project at the University of Massachusetts Amherst. As our lab was in the process of moving to Cornell, we sought to complete all work at Cornell University. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? No work was done on this project at the University of Massachusetts Amherst. As our lab was in the process of moving to Cornell, we sought to complete all work at Cornell University.

Publications