Filamentous phage derived biological nanorods : development of a novel display system : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Manawatū, New Zealand

Abstract

The Ff filamentous bacteriophage are filament-like bacterial viruses approximately 900 nm in length. The F-pilus-specific filamentous phage are resistant to heat, pH-extremes, and detergents in combination with their structural properties and amenability to DNA recombinant engineering has enabled their extensive use in modern biotechnology. However, the use of Ff-phage in vaccines and other such biological uses is controversial due to their ability to replicate in gut Escherichia coli, and the possibility of mobilisation and horizontal gene transfer of antibiotic resistance-encoding genes among the gut bacteria. As such, the novel system was established to create short, stable particles that cannot replicate, called NanoZap particles. However, this system has the disadvantage of often producing multiple-length particles, rather than the desired single-length particles; another disadvantage is that during packaging, one particle in a million packages the entire plasmid due to recombination that removes the terminator copy of the (+) ori, and given that these plasmids contain antibiotic resistance genes, this likely would spread antibiotic resistance throughout the surrounding environment. In this thesis several variations upon the original pNanoZap vector were created and tested to obtain monodisperse unit-length particles. The deletion of the complete multiple cloning site (MCS) that lied between the initiator and terminator of replication from the original pNanoZap vector achieved this aim. To eliminate rare antibiotic resistant particles that package the complete pNanoZap vector, the antibiotic resistance gene was removed from the pNanoZap 537 vector and replaced with an auxotrophic marker nadC; this vector was named pNanoZap 537N. It is yet to be seen if this new pNanoZap vector is capable of producing NanoZap particles.

For high-sensitivity diagnostics it is desirable to construct high-avidity particles, containing large number of detector molecules. To achieve this a double-display (detector displayed on phage which in turn is displayed on the surface of florescent E. coli) was designed and tested. When the E. coli expressing red fluorescent protein TinselPurple were infected with the bacteriophage, the chromogenic protein was lost, thereby showing that a different method of colouring E. coli will need to be used in order to construct the double-display particles.