Team:Imperial College/Modelling P3HBsyn

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Modelling: P(3HB) synthesis module

This page is about modelling the plastic-synthesising module developed in our project. The plastic that this module synthesises is the bioplastic poly-3-hydroxybutyrate, also known as P(3HB). The content below shows the process of building, designing and optimisation of the model. Also, the considerations, assumptions and limitations will be discussed.

Specifications

  • Input = monomer (3HB)
  • Output = polymer (P(3HB))

Deterministic model

The model was built and simulated in Simbiology, a Matlab package designed for modelling biological systems. In our model we have a compartment (labelled "E. coli_1") that represents our engineered cell. The compartment contains the reactions and species that interact with the polymer synthesis pathway.

The pathways on the right half of the cell are genetic expressions of the 3 enzymes involved in the plastic synthesis pathway. The pathways on the left represent the actual P(3HB) production pathway.

Imperial College Circuit.JPG Imperial College Circuit key.JPG

ODEs in Matlab Simbiology model

Genetic regulations and expressions

BDH2 (3-hydroxybutyrate dehydrogenase)

Alt text
Schematic of the gene expression of BDH2 as seen in the model diagram
Imperial College BDH2 gene.JPG

PhaB (Acetoacetyl-CoA reductase)and PhaC (P(3HB) synthase)

Alt text
Schematic of the gene expression of PhaB and PhaC as seen in the model diagram
Imperial College PhaBC gene.JPG

Values, sources and assumptions

Parameter Description Value Units Sources Assumptions
β maximum rate of transcription 0.032 mM/min
n Hill coefficient 2.0 dimensionless
K Activation coefficient 0.0031 mM
d_mRNA mRNA degradation rate 0.1 1/min
d_protein Protein degradation rate 0.050 1/min There is no active degradation pathway and that dilution is the dominant way by which the protein level decreases in a cell
k2 mRNA production rate 4.7 1/min
k3 Protein production rate 0.039 1/min
[Arabinose] Concentration of arabinose 0.008 mM
[mRNA] Concentration of mRNA - mM
[BDH2] Concentration of BDH2 - mM
[PhaB] Concentration of PhaB - mM
[PhaC] Concentration of PhaB - mM

Enzyme kinetics

BDH2 (3-hydroxybutyrate dehydrogenase)

atoAD (Acetyl-CoA:acetoacetyl-CoA transferase (α and β subunits))

phaB (Acetoacetyl-CoA reductase)

phaC (P(3HB) synthase)

Simulation results

P(3HB) production

Model-guided design and optimisation

Sensitivity analysis: species concentrations

What is sensitivity analysis and why do it?

In order to look at how we could increase the production of P(3HB) we decided to run a sensitivity analysis (also in Simbiology) to identify what species in the model P(3HB) is sensitive to, given the specific conditions with which we set the model. In other words, we calculated the time-dependent sensitivity of P(3HB) with respect to the initial conditions of other species (species formed along the synthesis pathway and enzymes involved).

Imperial College Half-normalised species.JPG

Scan with different levels of PhaB

The plot below is to show that increasing PhaB concentration within the engineered organism would have a positive effect on the concentration of the P(3HB) synthesised. As so many parameters within the model were taken from different sources, it would be difficult to give a simulation plot that is accurate quantitatively. Therefore, this plot should be interpreted from a qualitative perspective and the trend should be observed.

Imperial College PhaB scan Single cellmM.JPG

Difference between promoter expressions after optimisation

Metabolic considerations


The metabolic model predicts the interaction of our pathway with endogenous pathways. In synthetic biology, an ideal system is fully orthogonal. However, it can be the case, such as here that coenzymes are needed for the reaction steps in a metabolic pathway. When we realised that our pathway uses a lot of Co-A, we constructed a model to see how this can be recycled and predict whether it would negatively effect the chassis. Tell that the Co-A is regenerated at the same speed as it is used and therefore there is not a huge amount of Co-A "stuck" in the pathway at any one time. (Bobby, how much Co-A is that exactly?) This informed us about that the cells should be able to bear the burden of the pathway.

Metabolic model to optimise PHB production. Tell us how you predicted that phaB is the key enzyme and justify why the increase in enzyme level will increase the amount of PHB. This informed our design and we are changing the promoter in front of the phaCAB operon. Tell that if we can predict that it would be useful to downregulate any genes in the native metabolism of E.coli.


Effect on growth and interference with metabolism

Initial concentrations of metabolites

Our Sponsors

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