Bacterial detection using phage display for the differentiation of pre- and post-infection bacteriophages: A model M13 system
Abstract
Phage, or viruses that infect bacteria, are excellent for bacterial detection because they are host-specific and progeny phages are rapidly produced. Phage display is where a specific peptide capable of binding to an epitope is expressed on a phage coat protein. Phage can be used for the detection of foodborne pathogens if phage infection can be confirmed. The goal of this research was to demonstrate if progeny phage produced through infection and replication in the potentially pathogenic host bacteria can be differentiated from parent phage initially added to the sample using phage display. Proof-in-principle experiments were done using the minor coat protein PIII of the E. coli F' bacteriophage M13. A recombinant phage was constructed to contain two different copies of the PIII gene, or gIII: a constitutively-expressed wild-type gIII; and an inducible modified gIII that produced a heptamer capable of binding to streptavidin. Parent recombinant phage were propagated in an E. coli capable of repressing transcription of the second gIII , which resulted in only wild-type PIII production. Upon replication in a simulated wild-type-host bacterium, progeny phage also transcribed the modified copy of gIII and, subsequently, displayed the PIII with a streptavidin-binding site. Phage expressing the streptavidin binding site were isolated using streptavidin-coated Petri and microtiter plates, magnetic beads, and gravity-flow chromatographic columns. Phage titers and enzyme-linked immunosorbent assays (ELISAs) reflected the phages' ability to bind streptavidin-coated surfaces. Phage-binding assays revealed that a partially derepressed lac promoter caused an increased background signal due to the undesired incorporation of the fusion protein into the phage. Using the phage titer-based methods, a 50-fold difference in binding efficiency was observed between the parent and progeny phages when the phage had been amplified in a simulated wild-type host strain. This phage display methodology could be applied using existing lateral flow, biochip, or antibody-based assays for the detection of foodborne pathogens.
Degree
Ph.D.
Advisors
Applegate, Purdue University.
Subject Area
Food science|Microbiology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.