The goal of the SiphoPhagID project is to answer the question of how a virus uses its full set of genes to
multiply inside a host cell and to use this fundamental knowledge for rational synthetic engineering. To
accomplish this, we will study the bacterial virus SPP1, which infects the Gram-positive bacterium Bacillus
subtilis.
We will conduct a systematic phenotypic analysis of an SPP1 library of single-gene deletion mutants to
investigate the function of each gene individually. We will classify genes as essential, auxiliary or neutral
for SPP1 infection of host bacteria with diverse genetic backgrounds and under different conditions. Next,
we will use an array of methods to assign biochemical functions to these proteins and identify the viral
cycle steps to which they contribute. Our goal is to compile a comprehensive list of the functions
necessary for SPP1 to multiply and takeover the host bacterium's functions, converting it into a viral
factory. We will then use computer-controlled mini-bioreactors, hypermutator systems, and deep
sequencing to study SPP1 evolution in continuous culture, optimize SPP1 mutants, and map dispensable
genomic regions through mutational scanning.
Knowledge of the genes essential for virus multiplication will guide the engineering of a minimal SPP1
genome using synthetic biology. This will be achieved through sequential gene deletion whose deleterious
effects will be (partially) countered by adaptive laboratory evolution in bioreactors to enhance infection
performance. Phages with a minimal genome that cannot cause host cell lysis will be used to assert their
potential for expressing heterologous gene clusters at a high level from the ~300 copies of the SPP1
genome maintained in a long-lasting infected cell.
Collectively, this project aims to establish the complete set of biochemical functions required for the
sustainability of a bacterial virus, generating the fundamental knowledge for its rational synthetic
engineering.