Physics of Bacteriophage λ Infection

Bacteriophage λ infects bacteria by ejecting its single molecule of double-stranded DNA (dsDNA) into the host bacterial cell, while leaving its empty capsid outside. The dsDNA of phage λ is approximately 17,000 nm in length with a persistence length of 50 nm, while the capsid is only 30 nm in radius. The capsid of phage λ is therefore highly pressurized due to the crowding, bending, and the electrostatic repulsion of the dsDNA. We are interested in the dynamics of the genome ejection from the phage λ to the host cell, a process initially driven by the stored energy density (pressure) in the capsid. We will study this process in vitro by constructing an artificial system into which the phage λ dsDNA can eject.

The basic design of our experiment is to partition a chamber into two compartments separated by a synthetic membrane capable of incorporating phage λ receptor protein LamB. The phage λ ejects its dsDNA when it binds to LamB. The synthetic membrane is minimally permeable to salts or proteins, so it enables us to separately control each of the environments of the two compartments, one for the phage λ and one for genome ejection destination (mimic of host cells). We can then closely monitor the dynamics of dsDNA ejection in vitro by including fluorescence probes in the destination compartment. Cellular components can also affect the rate of dsDNA ejection, and those we are interested to test for are simple and multivalent ions, DNA binding proteins, and RNA polymerase. Ions can shield the electrostatic repulsion of the dsDNA in the capsid; thus, if included in the phage λ compartment, they will reduce the pressure inside the capsid. DNA binding proteins, if included in the destination compartment, can bind to the dsDNA as it enters. However, they are too large to diffuse into the phage capsid, so they create a mechanism for biased diffusion of dsDNA into the destination compartment. RNA polymerase is known to be a strong molecular motor; thus, if included in the destination compartment, it has the potential to pull in the dsDNA by mechanical force. We want to learn the roles of these components in the phage λ infection by studying them independently in vitro.

The full research proposal can be found here.

 

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