Difference between revisions of "Team:US AFRL CarrollHS/Model"
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Revision as of 18:07, 16 October 2018
Overview
The goal of the modeling for our project is to determine what ribosomal binding site (RBS) provides the optimal amount of CheZ expression, as too much CheZ can negatively impact the microbes chemotactic ability, but a high enough concentration is required to initiate chemotaxis. Although the modelling below uses arbitrary units and therefore only useful for determining relative amounts of CheZ produced, it provides a good idea of which RBS’s result in much too little protein expression. Additionally, in the future, we can measure the protein expression obtained with one of the RBS’s and then use the modeling to predict the absolute production that each of the other RBS’s would yield.
Method
We modeled effect of RBS strength on expression of CheZ, the protein that causes our engineered microbe to move. We used COPASI software for our modelling, and the differential equations we used are shown below. Data for relative RBS strength came from the iGEM registry (http://parts.igem.org/Ribosome_Binding_Sites/Prokaryotic/Constitutive/Community_Collection). The RBS strength was changed in the model by changing k1R3.
Reactions in Model
R1) (Transcription) DNA -> mRNA + DNA
R2) (Degradation of mRNA) mRNA -> mRNA0
R3) (Ribosome binding to mRNA) mRNA + ribo = mRNA_ribo
R4) (Translation) mRNA_ribo -> peptide + mRNA_ribo
R5) (Degradation of peptides) peptide -> peptide0
R6) (Maturation) peptide -> protein
R7) (Degradation of proteins) protein -> protein0
Variables
Km - Michaelis-Menten constant
VR1 - Max rate of reaction for reaction 1
k1RN - Rate constant for reaction N
k2RN - Rate constant for the reverse of reaction N
Km - Michaelis-Menten constant
Vc1 - Volume of the compartment
Equation (1) models transcription and uses Michaelis-Menten kinetics. The Law of Mass Action was used for Equations (2), (3), (5), and (6), which describe the rate of change of the concentrations of the peptide, protein, mRNA-ribosome complex, and ribosome respectively. Equations (4), (7), and (8) are also based on the Law of Mass Action and account for the degradation of mRNA, peptides, and the proteins respectively. The initial concentrations of DNA and ribosomes were set at 1, and all other initial concentrations were 0.
Results and Discussion
Fig 1. Levels of CheZ expression for RBS 29, 32, 33, 34, and 35
The results indicate that RBS 33 provides very little expression of CheZ, and is most likely not suitable for our purposes. RBS 34 and 35 (34 is covered by 35 in the graph) result in the greatest production of CheZ, and RBS 32 and 29 yield slightly less CheZ than RBS 34 and 35. Determining which of these three would be most suitable for our project would require experimentally determining the absolute amount of CheZ produced when using one of the RBS’s so that predictions concerning the absolute concentration of CheZ achieved with each one of the RBS’s could be made.