In order to produce PLPs, M13KO7 (produces all the phage proteins) and the phagemid (containing the toxin gene) needs to be integrated into the cell. During the process, if p5 takes M13KO7 it will construct a replicative phage and if it takes the phagemid it will produce PLPs.

Our project, KILL XYL will be a cure against the disease caused by the plant pathogen Xylella fastidiosa. One of our approaches is to engineered phage-like particles (PLPs), that will inject toxic genes into the bacterium. Phage systems, like M13, have been employed in biotechnological applications, most prominently in the identification and maturation of medically-relevant binding molecules through phage display ^[1].

However, production of phages as M13 have longterm consequences in E. coli metabolism^[2]. M13 has a unique lifecycle among phages. One of the crucial events of its multiplication is the packaging of its own DNA in the capsid. To do so, M13 uses it's protein 5 (p5) to target M13 origin of replication (M13ori). This step is crucial for the phage formation.

Moreover, we wanted that our phage became non-replicative to limit the spread of GMOs in the environment. That led to the creation of phage-like particles (PLPs). The design of these PLPs need the genome of M13 modified to stop the replication and the addition of a phagemid in the factory cell E.coli. Therefore, to create PLPs we use a modified M13 genome (M13KO7) that has several genes inserts in it M13ori and we integrate a phagemid with a M13ori.

Thanks to Jacques VAN HELDEN interview we put attention to the risk of recombination between M13KO7 and the phagemid. This question matter because we aim to create PLPs that aren't able to replicate and thus spread in the environment. Therefore, we test this possibility along our wetlab tests.

Here we wanted to answer a single question: As p5 recognized M13ori, will the bacterium produces only PLPs or will it also produces replicative phages ?.

First iteration of the model

We started with an excessively simple model to see whether our understanding of our system was profound enough to allow room for such simplifications. We made the following assumptions in this spirit:

Which led to the following model:

Second iteration

This time, we decided to adopt the model of a "wild-type" M13's biology ^[2][3] to fit our own engineered version of M13 and better inform wet lab experiments, as well as explain some of their preliminary findings!

Model validation

compare particle profile to that obtained in wet lab

--> Insufficient p3

could cause the packing of several DNA molecules per phage.

What is the ideal number of plasmid copies

Wet-lab and dry-lab back and forth

References

1. ↑ Salmond, G. P. C. & Fineran, P. C. A century of the phage: past, present and future. Nat Rev Micro 13, 777–786 (2015).
2. ↑ ^{2.0 2.1} Smeal et al, Simulation of the M13 life cycle I: Assembly of a genetically-structured deterministic chemical kinetic simulation, Virology, 500, January 2017
3. ↑ Smeal et al, Simulation of the M13 life cycle II: Investigation of the control mechanisms of M13 infection and establishment of the carrier state, Virology, 500, January 2017