Team:UCSC/Phage Infection

Phage Infection

What are Bacteriophages?

Bacteriophages (phages) are viruses that selectively infect bacteria. Phages transport their genome from cell-to-cell using protein-based particles known as capsids. Upon infection, specialized proteins on the capsid inject viral genetic material into a host cell. The genetic material is then expressed, allowing it to hijack the host’s cellular machinery to replicate its own genome, package the genome into more capsids, and release them into the environment.

How Did We Use Them?

In our project, we take advantage of a phage’s ability to package and transport genes between bacterial cells while avoiding real viral infection. For the initial delivery to a target population, we use a modified form of the bacteriophage M13 to package and transport the plasmid encoding our gene-elimination mechanism. M13 phage is a filamentous, single-stranded DNA virus that infects E. coli that possess pili [2]. We plan to use this phage because we expect that it can infect both the DH5α F’-mcherry E. coli in our mcherry model and E. coli O157:H7 for our future application, as they both have F or F-like pili.

M13 bacteriophage virion binds to E. coli with F or F-like pilus, then inserts the circular single-stranded DNA genome it carries.

Creating Our Pseudovirion Delivery Vector

The construction of the pseudovirion delivery vector requires two different plasmids to be inside of a cell: helper phage and a phagemid. The helper phage plasmid encodes for the capsid structural and packaging proteins, but contains a damaged phage origin of replication (ori), so the helper phage cannot be packaged into viral particles[2]. Viral packaging proteins package plasmids that contain a phage ori, making a phagemid that contains a phage ori and desirable cargo [1]; in our case, the phagemid encodes the F1 ori and our gene-elimination system. The plasmid map below details the components of our phagemid design.

Plasmid maps depicting the genes for the M13KO7 helper phage (left) and φMINT phagemid (right) used to make the bacteriophage delivery system in the mcherry model.

Diagram depicts the phagemid-packaging mechanism. Phagemid (left) is packaged into the M13 capsid produced by the Helper Phage plasmid (right). Pseudovirion exits the cell into the surrounding media without lysing it.

Creating the Viral Delivery System

To construct the viral delivery system containing our synthetic genes, we will transform DH5α F’ cells with our phagemid and then infect them with M13KO7 helper phage. We will concentrate the viral particles secreted from helper phage-containing cells to then infect and deliver our gene disruption mechanism to our target E. coli.