Difference between revisions of "Team:Aix-Marseille/Model2"

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Our [[Team:Aix-Marseille/HP/Interviews|interviews]] and [[Team:Aix-Marseille/Legislation|legislation]] study  
 
Our [[Team:Aix-Marseille/HP/Interviews|interviews]] and [[Team:Aix-Marseille/Legislation|legislation]] study  
 
both showed that the number of viable phages could be a problem.
 
both showed that the number of viable phages could be a problem.
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We therefore decided to [[Team:Aix-Marseille/M13_test|measure]] and model the ratio between viable phage and phage-like particles,
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We therefore decided to [[Team:Aix-Marseille/M13_test|measure]] and model the ratio between viable phage and phage-like particles, and so try to optimize this ratio to facilitate the preparation of pure PLP.
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and so try to optimize this ratio to facilitate the preparation of pure PLP.
+
  
 
'''The Model'''
 
'''The Model'''
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<!-- Erwan check this and fix it -->
 
<!-- Erwan check this and fix it -->
 
This model was modified to incorporate:  
 
This model was modified to incorporate:  
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the helper-phage E. coli plasmid origin;
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The helper-phage E. coli plasmid origin;
 
the modification of the helper-phage M13 origin;
 
the modification of the helper-phage M13 origin;
 
the presence of a phagemid, with its own replicative origin and M13 origin.
 
the presence of a phagemid, with its own replicative origin and M13 origin.
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These modifications added xxx species, equations and yyy parameters to the original model.
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These modifications added xxx species, zzz equations and yyy parameters to the original model, and they modified www parameters of the original model.
 
The modified model is available [[File:T--Aix-Marseille--Model.zip|here.]] <!--it needs to be uploaded onto the iGEM server-->.
 
The modified model is available [[File:T--Aix-Marseille--Model.zip|here.]] <!--it needs to be uploaded onto the iGEM server-->.
  
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'''Results'''
 
'''Results'''
  
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We need one or two graphs here showing how the packaging ratio depends on different parameters.
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We need one or two graphs here showing how the packaging ratio depends on three main parameters: the DNA III polymerase binding rate to the E-coli origin of replication (Figure 2) of either the plasmid or phagemid, the initial ratio of transfected phagemid and helper phage (Figure 3), and the difference in p5 affinity for eitheir plasmid's M13 ori (Figure 4).
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We also observed increasing the number of transfected plasmids increased production, up to a certain point, which we determined (Figure 5).
 +
 
 
A description of these graphs.
 
A description of these graphs.
  
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<!--What we write here determines if you get a gold medal or not-->
 
<!--What we write here determines if you get a gold medal or not-->
 
Clearly the initial design needs to be improved to increase the proportion of PLP produced.
 
Clearly the initial design needs to be improved to increase the proportion of PLP produced.
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The model has shown as that the parameters of prime importance for determining the proportion of PLP produced are .... <!--This corresponds to the figures-->
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The model has shown as that the parameters of prime importance for determining the proportion of PLP produced are .... <!--This corresponds to the figures 2 through 5.
 
So to implement our project it will be necessary to redesign certain features. Specifically <!-- What goes here -->.
 
So to implement our project it will be necessary to redesign certain features. Specifically <!-- What goes here -->.

Revision as of 14:26, 1 November 2017

Modelling PLP production

To produce PLPs, bacteria must contain both a helper phage (like M13KO7), that codes for the different phage proteins, and also a phagemid, that encodes the toxic gene we want to include in the PLP. If M13KO7 is packaged a replicative phage is produced, which we do not want, and if the phagemid it packaged then a PLP is produced.

The problem

Our objective is to produce phage like particles (PLP), for this, the bacteria must contain both a helper phage and also a phagemid (see figure). During phage production, several key events determine which DNA molecules are packaged into phage or PLP. These involve recognition of the M13 replication origin by several phage proteins.

A major hurdle to marketing KILL XYL is obtaining the necessary authorizations. Our interviews and legislation study both showed that the number of viable phages could be a problem. We therefore decided to measure and model the ratio between viable phage and phage-like particles, and so try to optimize this ratio to facilitate the preparation of pure PLP.

The Model

We based our model on a recently published model of "wild-type" M13 replication [1] [2]. This model was modified to incorporate: The helper-phage E. coli plasmid origin; the modification of the helper-phage M13 origin; the presence of a phagemid, with its own replicative origin and M13 origin. These modifications added xxx species, zzz equations and yyy parameters to the original model, and they modified www parameters of the original model. The modified model is available File:T--Aix-Marseille--Model.zip .

We used the initial measurement to constrain parameters of the model, along with published numbers for the number of copies of plasmids with different replicative origins.

Results

We need one or two graphs here showing how the packaging ratio depends on three main parameters: the DNA III polymerase binding rate to the E-coli origin of replication (Figure 2) of either the plasmid or phagemid, the initial ratio of transfected phagemid and helper phage (Figure 3), and the difference in p5 affinity for eitheir plasmid's M13 ori (Figure 4). We also observed increasing the number of transfected plasmids increased production, up to a certain point, which we determined (Figure 5).

A description of these graphs.

Conclusions

Clearly the initial design needs to be improved to increase the proportion of PLP produced.

The model has shown as that the parameters of prime importance for determining the proportion of PLP produced are .... .
  1. ↑ Smeal et al, Simulation of the M13 life cycle I: Assembly of a genetically-structured deterministic chemical kinetic simulation, Virology, 500, January 2017
  2. ↑ 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