Monomeric streptavidin phage display allows efficient immobilization of bacteriophages on magnetic particles for the capture, separation, and detection of bacteria.

Immobilization of bacteriophages onto solid supports such as magnetic particles has demonstrated ultralow detection limits as biosensors for the separation and detection of their host bacteria. While the potential impact of magnetized phages is high, the current methods of immobilization are either weak, costly, inefficient, or laborious making them less viable for commercialization. In order to bridge this gap, we have developed a highly efficient, site-specific, and low-cost method to immobilize bacteriophages onto solid supports. While streptavidin-biotin represents an ideal conjugation method, the functionalization of magnetic particles with streptavidin requires square meters of coverage and therefore is not amenable to a low-cost assay. Here, we genetically engineered bacteriophages to allow synthesis of a monomeric streptavidin during infection of the bacterial host. The monomeric streptavidin was fused to a capsid protein (Hoc) to allow site-specific self-assembly of up to 155 fusion proteins per capsid. Biotin coated magnetic nanoparticles were functionalized with mSA-Hoc T4 phage demonstrated in an E. coli detection assay with a limit of detection of < 10 CFU in 100 mLs of water. This work highlights the creation of genetically modified bacteriophages with a novel capsid modification, expanding the potential for bacteriophage functionalized biotechnologies.

Enterobacteria Phage SV76 Host Range and Genomic Characterization.

Background: Increasing the quantity and detail of bacteriophage genomic data is critical to broadening our understanding of how bacteriophages operate to allow us to harness their unique properties for biotechnology advancements. Here we present the complete sequence of phage SV76's assembled and annotated genome (Accession OM339528). SV76 has previously been classified as a T4-like bacteriophage belonging to the Tequatrovirus genus within the Myoviridae family of contractile tailed bacteriophages. Materials and Methods: Whole genome sequencing, assembly, and annotation was performed on SV76. Double-agar spot assays were utilized to determine SV76's host range against a panel of 72 Escherichia coli isolates meant to represent the diversity of E. coli, as well as a series of knockouts designed to identify required receptor binding proteins. The genome and host range were compared to the closely related phage, T2. Results: Spot assays revealed that SV76 could plaque on 10 of the 72 strains (13.9 %) and nine of the nine E. coli K12 single gene knockout of known phage receptors (100%). SV76 did not plate on a ΔfadL E. coli indicating suggesting a requirement as a receptor binding protein. Conclusions: SV76 is closely related to T2 with similar host ranges within ECOR. This study presents novel host range and genomic data on SV76 phage, providing a foundation for future studies to further characterize SV76 to understand more about SV76 and other T4-like phages that can be applied to create novel biotechnologies.

Bacteriophage Capsid Modification by Genetic and Chemical Methods.

Bacteriophages are viruses whose ubiquity in nature and remarkable specificity to their host bacteria enable an impressive and growing field of tunable biotechnologies in agriculture and public health. Bacteriophage capsids, which house and protect their nucleic acids, have been modified with a range of functionalities (e.g., fluorophores, nanoparticles, antigens, drugs) to suit their final application. Functional groups naturally present on bacteriophage capsids can be used for electrostatic adsorption or bioconjugation, but their impermanence and poor specificity can lead to inconsistencies in coverage and function. To overcome these limitations, researchers have explored both genetic and chemical modifications to enable strong, specific bonds between phage capsids and their target conjugates. Genetic modification methods involve introducing genes for alternative amino acids, peptides, or protein sequences into either the bacteriophage genomes or capsid genes on host plasmids to facilitate recombinant phage generation. Chemical modification methods rely on reacting functional groups present on the capsid with activated conjugates under the appropriate solution pH and salt conditions. This review surveys the current state-of-the-art in both genetic and chemical bacteriophage capsid modification methodologies, identifies major strengths and weaknesses of methods, and discusses areas of research needed to propel bacteriophage technology in development of biosensors, vaccines, therapeutics, and nanocarriers.

Optimization of T4 phage engineering via CRISPR/Cas9.

A major limitation hindering the widespread use of synthetic phages in medical and industrial settings is the lack of an efficient phage-engineering platform. Classical T4 phage engineering and several newly proposed methods are often inefficient and time consuming and consequently, only able to produce an inconsistent range of genomic editing rates between 0.03-3%. Here, we review and present new understandings of the CRISPR/Cas9 assisted genome engineering technique that significantly improves the genomic editing rate of T4 phages. Our results indicate that crRNAs selection is a major rate limiting factor in T4 phage engineering via CRISPR/Cas9. We were able to achieve an editing rate of > 99% for multiple genes that functionalizes the phages for further applications. We envision that this improved phage-engineering platform will accelerate the fields of individualized phage therapy, biocontrol, and rapid diagnostics.

Phage-based biosensors: in vivo analysis of native T4 phage promoters to enhance reporter enzyme expression.

Phage-based biosensors have shown significant promise in meeting the present needs of the food and agricultural industries due to a combination of sufficient portability, speed, ease of use, sensitivity, and low production cost. Although current phage-based methods do not meet the bacteria detection limit imposed by the EPA, FDA, and USDA, a better understanding of phage genetics can significantly increase their sensitivity as biosensors. In the current study, the signal sensitivity of a T4 phage-based detection system was improved via transcriptional upregulation of the reporter enzyme Nanoluc luciferase (Nluc). An efficient platform to evaluate the promoter activity of reporter T4 phages was developed. The ability to upregulate Nluc within T4 phages was evaluated using 15 native T4 promoters. Data indicates a six-fold increase in reporter enzyme signal from integration of the selected promoters. Collectively, this work demonstrates that fine tuning the expression of reporter enzymes such as Nluc through optimization of transcription can significantly reduce the limits of detection.