Team:Bielefeld-Germany/Modell
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Figure 2 shows the simulation of our model for a timespan of 120 minutes. | Figure 2 shows the simulation of our model for a timespan of 120 minutes. | ||
- | [[Image:Bielefeld-Germany2011-E500bos250k139k2017.png|center| | + | [[Image:Bielefeld-Germany2011-E500bos250k139k2017.png|center|800px|thumb|'''Fig. 2: Simulation of extracellular degradation of BPA in combination with the activation of the molecular beacon. ''']] |
=Modelling of intracellular bisphenol A degradation= | =Modelling of intracellular bisphenol A degradation= |
Revision as of 16:22, 20 September 2011
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Contents |
Modelling of cell-free bisphenol A degradation
In our project we want to construct a cell-free biosensor that can detect bisphenol A (BPA). To be more specific we want to fuse proteins to silicia beads that degrade the BPA and detect the NAD+ that is set free in the reaction by a NAD+-dependent ligase (also attached to the beads) in combination with molecular beacons.
First reaction
The first reaction is the degradation of BPA by the Cytochrome P450 monooxygenase system (the components of this system are cytochrome P450 (<partinfo>K123000</partinfo>), ferredoxin (<partinfo>K123001</partinfo>) and ferredoxin reductase (<partinfo>K525499</partinfo>)). The velocity of the BPA degradation can be calculated by Michaelis Menten enzyme kinetics:
The velocity V1 was calculated in dependency of the enzyme concentration and the kcat-value for the monooxygenase system of [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1317416/ 3.9 min-1].
Second reaction
In the second reaction the NAD+-dependent DNA-ligase LigA from E.coli (<partinfo>K525710</partinfo>) is used to transfer the molecular beacons from the closed state bcs to the open state bos. It is described by the following reaction scheme:
The rate of a reaction with two substrates is calculated by the following equation (assuming initial product concentration of 0):
where KiA is the binding affinity of the inhibitor to substrate A, KmA and KmB the Michaelis-Menten constants for substrate A respectively B in the specific reaction. So the amount of the molecular beacon open state is described by the equation
For this we assume, that the molecular amount of NAD+ is equal to the molecular amount of degraded BPA. The required electrones for the degradation of one BPA molecule, more precisely for the activation of the oxygen in the cytochrome P450 monooxygenase system, are released by the oxidation of one molecule of the cofactor NADH.
The velocity V2 is calculated by multiplicating the kcat-value of 0.0212 s-1 found in the [http://onlinelibrary.wiley.com/doi/10.1046/j.1432-1327.2000.01377.x/pdf literature] with the used concentration of NAD+. For KmNAD+ and Kmbos we use the values we found in the literature ([http://www.sciencedirect.com/science/article/pii/S0003269702003020 Chen et al., 2002]):
KmNAD+= 0.5 µM
Kmbos= 0.06 µM
Regarding KiNAD+ we considered AMP and NMN as possible inhibitors ([http://www.sciencedirect.com/science/article/pii/S0003269702003020 Chenet al., 2002]) for the reaction. The literature provides half maximal inhibitory concentrations (IC50-values) of 3.1 mM for AMP and 9.5 for NMN µM. We assume that AMP has no significant influence in our model, as the IC50-value is quite high. The IC50-value of NMN on the other hand is in closer range to concentrations that migth be achieved during the performance of our test, so we do consider it in our calculations. To determine KiNAD+ we used the formula
yielding an value of 0.45 µM for NMN.
Implementation of the equations for the first and the second reaction in matlab:
Figure 2 shows the simulation of our model for a timespan of 120 minutes.
Modelling of intracellular bisphenol A degradation
The modelling was done with the software [http://www.berkeleymadonna.com/ Berkeley Madonna] using the [http://en.wikipedia.org/wiki/Runge–Kutta_methods#Common_fourth-order_Runge.E2.80.93Kutta_method common fourth-order Runge-Kutta] method to solve the equations. The model was fitted to the measured data by the function "curve fit" in Berkeley Madonna to calculate the parameters, constants etc.
To model the BPA degradation by E. coli carrying BioBricks for BPA degradation (<partinfo>K123000</partinfo> and <partinfo>K123001</partinfo>) the cell growth has to be described first. The observed growth of E. coli on (our) LB medium was [http://en.wikipedia.org/wiki/Diauxie diauxic] with two different growth phases. Cell growth is a [http://en.wikipedia.org/wiki/First_order_kinetics#First-order_reactions first-order reaction] and is mathematically described as
with the specific growth rate µ and the cell count X. The specific growth rate is dependent on the concentration of the growth limiting substrate (e.g. glucose) and can be described as
with the substrate concentration S, the Monod constant KS and the maximal specific growth rate µmax ([http://www.annualreviews.org/doi/abs/10.1146/annurev.mi.03.100149.002103 Monod, 1949]). Because LB medium is a complex medium we cannot measure the substrate concentration so we have to assume an imaginary substrate concentration. Due to the diauxic growth two different substrates with different Monod constants and consumption rates are necessary to model the cell growth. The amount of a substrate S can be modelled as follows
with the specific substrate consumption rate per cell qS. The whole model for the diauxic growth of E. coli on LB medium with two not measurable (imaginary) substrates looks like:
The specific BPA degradation rate per cell qD is modelled with an equation like eq. (3). In the beginning of the cultivations, when E. coli growths on the "good" imaginary substrate S1, no BPA degradation is observed. When this substrate is consumed, the BPA degradation starts. The model for this diauxic behavior is as follows:
Fig. 1 shows a comparison between modelled and measured data for cultivations with BPA degrading E. coli. In Tab. 1 the parameters for the model are given obtained by curve fitting the model to the data.
![](/wiki/images/7/73/Bielefeld-Germany2011-BPAdegradEcoliModel.jpg)
Tab. 1: Parameters of the model.
Parameter | K525512 | K525517 |
---|---|---|
X0 | 0.112 108 mL-1 | 0.138 108 mL-1 |
µmax | 1.253 h-1 | 1.357 h-1 |
KS,1 | 2.646 AU-1 | 1.92 AU-1 |
KS,2 | 265.1 AU-1 | 103.1 AU-1 |
S1,0 | 1.688 AU | 1.166 AU |
qS,1 | 0.478 AU 10-8 cell-1 | 0.319 AU 10-8 cell-1 |
S2,0 | 16.091 AU | 6.574 AU |
qS,2 | 0.295 AU 10-8 cell-1 | 0.191 AU 10-8 cell-1 |
BPA0 | 0.53 mM | 0.53 mM |
qD | 8.76 10-11 mM cell-1 | 1.29 10-10 mM cell-1 |
The specific BPA degradation rate per cell qD is about 50 % higher when using the fusion protein compared to the polycistronic bisdAB gene. This results in an average 9 hours faster, complete BPA degradation by E. coli carrying <partinfo>K525517</partinfo> compared to <partinfo>K525512</partinfo> as observed during our cultivations. The fusion protein between BisdA and BisdB improves the BPA degradation by E. coli.
References
Sasaki M, Akahira A, Oshiman K, Tsuchido T, Matsumura Y (2005b) Purification of Cytochrome P450 and Ferredoxin, Involved in Bisphenol A Degradation, from Sphingomonas sp. Strain AO1, [http://aem.asm.org/cgi/content/abstract/71/12/8024 Appl Environ Microbiol 71(12):8024-8030].
Georlette D, Jonsson Z, Van Petegem F, Chessa J-P, Van Beeumen J, Huebscher U, Gerday C (2000) A DNA ligase from psychrophile Pseudoalteromonas haloplanktis gives insigth into the adaption of proteins to low temperatures, [http://onlinelibrary.wiley.com/doi/10.1046/j.1432-1327.2000.01377.x/pdf Eur.J.Biochem 267:3502-3512].
Chen X, Hentz N, Hubbard F, Meier T, Sittampalam S, Zhao G (2002) Development of a fluorescence resonance energy transfer assay for measuring the activity of Streptococcus pneumoniae DNA ligase, an enzyme essential for DNA replication, repair and recombination, [http://www.sciencedirect.com/science/article/pii/S0003269702003020 Anal Biochem 309:232-240].
Monod J (1949) The growth of bacterial cultures, Annu Rev Microbiol [http://www.annualreviews.org/doi/abs/10.1146/annurev.mi.03.100149.002103 3:371-394].