Designing of the biosynthesis reactions of Pyoverdine.
Found an article with full description about the Pyoverdine of P. putida KT2440, structure formule
is also included. Divided the biosynthesis of pyoverdine in 8 reactions, included the modified amino acids and added these to the model. The pathway can be found in pathways and metabolites.
Pyoverdine added to the GSMM. The next step was to check the parameters for the production of Pyoverdine. The Pyoverdine production was tested against nitrogen uptake, glucose uptake and biomass production. These fluxes were tested in a 4D plot to check its dependency's. The next thing was validating these maximum production fluxes against data from in vitro experiments to get a reference. One article can be found about the production kinetics of pyoverdine production but the dimension are not usable. So these experiments have to be performed here in the lab. So After validation and changing of my experiment work started on the next fungal growth inhibitor, that was the metabolite DAPG. The biosynthesis pathway was found in literature. So these have were added to the model divided in 4 reactions . pathways and metabolites
Mainly focusing on executing the experiments that were setup last week.
the production values of DAPG are tested against the nitrogen uptake and sulfate uptake to take a
reference against the metabolites that have an increased requirement of these elements. As
expected the change of the uptakes did not change the production of DAPG as you expect from the
structure formule. Next thing to do is writing a script that tests the DAPG production against the total production pool of CoA to see if there is a big change in the amount of CoA needed correlated with growth rate.
For further details about this experiment and the results check the Lab notes and fungal growth inhibitors. In the wait steps of this experiment the analysing continued with the DAPG production kinetics in the extended model and the results for the metabolic stress that is induced by the fungal growth inhibitor.
Writing report for the toolbox. In this toolbox the experiment is included with the title: Determination of pyoverdin production kinetics by P. Putida KT2440.
Focused on adding proteins to the model. A test was performed to check for differences if proteins are added with their protein sequences and proteins described as in the model for biomass production. Conclusion is that is does not have a great impact on the growth rate. Still choose for adding with the protein sequences for being as accurate as possible.
This week all the toxins and Kill-Switch are combined in one model. For the toxins: Pyoverdin and Pyocin the production kinetics are known as well as for the Kill-Switch (production value calculated from input model design) for the other 2 toxins there is still a lot of literature search needed to get correct production values. I have chosen for an irreversible model to be able to minimize the total flux trough the model, to get rid of existing loops in a Flux balance Analysis (FBA).
The Toxin/anti-Toxin part of our system is added to the model. A literature research has been done to find all the remaining production rates. The new model is tested and improved to run the simulation faster. Multiple carbon sources is now possible and it is now possible to change the oxygen uptake rate.
All the simulations have run this week. Simulations have run for glucose and the different carbon compositions. Other important simulation was the parameter analysing. This is performed all parameters combined and separately. A last simulation was performed for two variable glucose and oxygen but it has failed so far because of the forces uptake in certain ratio's (wrong ratio gives infeasible FBA results).
The simulation with glucose and oxygen as variable could not run properly in minimized flux model. This has been changed back to a normal model. This could cause minor differences in the results but was necessary to perform this simulation.
The only thing left to do was making the plots for the wiki’s. This is a time consuming process. In the waiting time. I focused on writing the metabolic modeling part.
"
