Difference between revisions of "Team:NCKU Tainan/Kinetic Law"
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D is the diffusion coefficient or diffusivity | D is the diffusion coefficient or diffusivity | ||
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| − | <p class="pcontent">After | + | <p class="pcontent">After CO<sub>2</sub> diffused into cell, it will have a reversible reaction.</p> |
<p class="pcontent">$${CO_2 + H_2O \underset{k_{-1}}{\stackrel{k_1}{\rightleftharpoons}} H^+ + HCO_3^-}$$</p> | <p class="pcontent">$${CO_2 + H_2O \underset{k_{-1}}{\stackrel{k_1}{\rightleftharpoons}} H^+ + HCO_3^-}$$</p> | ||
<p class="pcontent">That’s what CA catalyze the reversible interconversion of CO<sub>2</sub> and HCO<sub>3</sub><sup>-</sup>. | <p class="pcontent">That’s what CA catalyze the reversible interconversion of CO<sub>2</sub> and HCO<sub>3</sub><sup>-</sup>. | ||
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<div id="centerimg"> | <div id="centerimg"> | ||
| − | <img class="twoimg contentimg" src=" | + | <img class="twoimg contentimg" src="https://static.igem.org/mediawiki/2018/9/9f/T--NCKU_Tainan--kinetic_law_fig1.jpg"> |
<p class="pcontent">Fig. 1 CO<sub>2</sub> uptake model diagram of simbiology</p> | <p class="pcontent">Fig. 1 CO<sub>2</sub> uptake model diagram of simbiology</p> | ||
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| − | <img class="twoimg contentimg" src=" | + | <img class="twoimg contentimg" src="https://static.igem.org/mediawiki/2018/8/83/T--NCKU_Tainan--kinetic_law_fig3.png"> |
<p class="pcontent">Fig. 3 Original metabolism model diagram of simbiology</p> | <p class="pcontent">Fig. 3 Original metabolism model diagram of simbiology</p> | ||
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| − | <img class="twoimg contentimg" src=" | + | <img class="twoimg contentimg" src="https://static.igem.org/mediawiki/2018/4/41/T--NCKU_Tainan--kinetic_law_fig4.png"> |
<p class="pcontent">Fig. 4 TCA cycle model diagram of simbiology</p> | <p class="pcontent">Fig. 4 TCA cycle model diagram of simbiology</p> | ||
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| − | <img class="twoimg contentimg" src=" | + | <img class="twoimg contentimg" src="https://static.igem.org/mediawiki/2018/a/aa/T--NCKU_Tainan--kinetic_law_fig5.png"> |
<p class="pcontent">Fig. 5 TCA cycle model diagram of simbiology</p> | <p class="pcontent">Fig. 5 TCA cycle model diagram of simbiology</p> | ||
</div> | </div> | ||
<div id="centerimg"> | <div id="centerimg"> | ||
| − | <img class="twoimg contentimg" src=" | + | <img class="twoimg contentimg" src="https://static.igem.org/mediawiki/2018/e/e6/T--NCKU_Tainan--kinetic_law_fig6.png"> |
<p class="pcontent">Fig. 6 whole xylose metabolisml diagram of simbiology</p> | <p class="pcontent">Fig. 6 whole xylose metabolisml diagram of simbiology</p> | ||
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Revision as of 13:26, 24 September 2018
Kinetic law
Kinetic law of Modeling
Mathematic model helps us to predict the reaction result before experiment. First of all, we have to define which kinetic law can be applied into which metabolic pathway and chemical equation. There are three kinds of kinetic we chose in this model, mass action, Henri-Michaelis-Menten, and Ping-Pong-Bi-Bi.
- Mass action
- Henri-Michaelis-Menten
- Ping-Pong-Bi-Bi
In chemistry, the law of mass action is the proposition that the rate of a chemical reaction is directly proportional to the product of the activities or concentrations of the reactants. Mass action kinetics describes this behavior as an equation where the velocity or rate of a chemical reaction is directly proportional to the concentration of the reactants.
$${aA + bB \xrightarrow{Kf} cC + dD}$$
$${aA + bB \xrightarrow{Kr} cC + dD}$$
$${Kf = {[C]^c[D]^d \over [A]^a[B]^b}}$$
$${Kr = {[A]^a[B]^b \over [C]^c[D]^d}}$$
Where Kf is forward rate of equilibrium, Kr is reverse rate of equilibrium
In 1913, Michaelis and Menten studied the invertase system and found some behavioral patterns of enzyme-matrix reaction. When the enzyme is catalyzed, the matrix will be combined with the enzyme to generate the product. However, the binding of the enzyme to the matrix is reversible. The concentration of ES will be constant when the reaction is steady state. They also proposed the mathematical description of the behavior of the enzyme. They can express the relationship between the reaction rate and the enzyme mathematically. Hence, the reaction rate is in a hyperbolic relationship with the concentration of substrate at a fixed amount of enzyme. That is the Michaelis-Menten kinetic law.
A. The Henri-Michaelis-Menten kinetic law is expressed as follows:
$${E + S \underset{k_{-1}}{\stackrel{k_1}{\rightleftharpoons}} ES \xrightarrow{k_2} E + P}$$
The Enzyme(E) forms a Complex (ES) with the Substrate (S) and modifies it to the Product (P). The Product is then released and the Enzyme is free to perform another modification. This process is described by the ODE system.
The system cannot be solved analytically. Michaelis and Menten considered a quasi equilibrium between E and the ES-Complex to simplify the system.
$${{dS \over dt} = -k_1E \cdot S + k_{-1}ES}$$
$${{dES \over dt} = k_1E \cdot S - (k_{-1} + k_2)ES}$$
$${{dE \over dt} = k_1E \cdot S - (k_{-1} + k_2)ES}$$
$${{dP \over dt} = k_2ES}$$
After derivation $${v = {-ds \over dt} = {dP \over dt}}$$
where Vm represents the maximum rate achieved by the system, and the Km is the substrate concentration at which the reaction rate is half of Vm.
The constants vmax and Km can be looked up at the public data base BRENDA. The rate konstant k2 describes how many substrate molecules are transformed into product molecules per second and active site, it is thus called turnover number.
$${Vmax = k_2 \cdot E_{total} = k_{cat} \cdot E_{total}}$$
B. A more realistic description of a enzymatic reactions than pure Michaelis Menten Kinetics is given by considering the product forming reaction step as reversible.
$${E + S \underset{k_{-1}}{\stackrel{k_1}{\rightleftharpoons}} ES \xrightarrow{k_2} E + P}$$
The Enzyme(E) forms a Complex (ES) with the Substrate (S) and modifies it to the Product (P). In the reversible Michaelis-Menten reaction the Product can bind the enzyme again and react back to the substrate. This process is described by the ODE system.
$${{dS \over dt} = -k_1E \cdot S + k_{-1}ES}$$
$${{dES \over dt} = k_1E \cdot S + k_{-2}E \cdot P - (k_{-1} + k_2)ES}$$
$${{dE \over dt} = k_1E \cdot S - k_{-2}E \cdot P - (k_{-1} + k_2)ES}$$
$${v = {dP \over dt} = k_2ES - k_{-2}E \cdot P = v_f - v_b}$$
After derivation $${v = {-ds \over dt} = {dP \over dt}}$$
Finally we use Vfmax=k2⋅Etotal and Vbmax=k−1⋅Etotal to get the common form for the reversible Michaelis Menten equation
$${v = {v^{max}_f S/K_{m,1} - v^{max}_b P/K_{m,2} \over (1 + S/K_{m,1} + P/K_{m,1})}}$$
Where Vf is the forward rate and the Vb is backwared rate while $${K_{m,1} = {K_{-1} + K_2 \over K_1}}$$ or represents as KS, and $${K_{m,2} = {K_{-1} + K_2 \over K_{-2}}}$$ or represent as KP.
For Ping-Pong-Bi-Bi, it is a double displacement reactions. Hence, there is more than one matrix in the reaction, and there may be several products for multi-matrix reactions in most of enzyme reactions. For example, S1 + S2 → P1 +P2. Binding of A and B are the different enzyme forms, E and F respectively. The double matrix reaction can still be applied to the Henri-Michaelis-Menten formula, but the Km of the two matrices should be determined separately. In addition, the concentration of S2 in the reaction should be saturated when S1 is measured.
Method of our Modeling
We choose the simbiology in matlab to develop our model. SimBiology provides an app and programmatic tools to model, simulate, and analyze dynamic systems which can easily applied on systems biology.
Pathway of our project
Part A : (CO2 uptake model)
CO2 diffuse into cell can use Fick’s law.
$${[CO_2]_{air} \rightarrow [CO_2]_{uptake}}$$
Assume E coli was a sphere with 10-6 radius while the depth of membrane was 4*10-9
$${J = {-D{dφ \over dx}}}$$
J is the "diffusion flux," of which the dimension is amount of substance per unit area per unit time. D is the diffusion coefficient or diffusivity
After CO2 diffused into cell, it will have a reversible reaction.
$${CO_2 + H_2O \underset{k_{-1}}{\stackrel{k_1}{\rightleftharpoons}} H^+ + HCO_3^-}$$
That’s what CA catalyze the reversible interconversion of CO2 and HCO3-. Although some CAs prefer the direction of CO2 hydration, the carboxysomal CAs in cyanobacteria and some chemoautotrophic bacteria favor the direction of HCO3− dehydration. The result of model and our experiments showed that CA improve amount of CO2 uptake and CO2 utilization.
Fig. 1 CO2 uptake model diagram of simbiology
Part B : (Xylose – PRK – Rubisco model)
We clone our genes into E.coli to make it fix the carbon dioxide. The genes include PRK, Rubisco. On the other hand, we also want to produce the higher value product in our project.
| Equation | Enzyme | Kinetic law |
|---|---|---|
| $${Xylose \rightarrow Xylulose}$$ $${Xylulose + ATP \rightarrow X5P}$$ $${X5P \rightarrow Ru5P}$$ | xylose isomerase | Henri-Michaelis-Menten |
| $${Ru5P \xrightarrow{PRK} + RuBP}$$ | Phosphoribulokinase | Henri-Michaelis-Menten |
| $${RuBP + CO_2 \xrightarrow{RuBisCo} + 3PG}$$ | Ribulose-1,5-bisphosphate carboxylase/ oxygenase | Ping-Pong-Bi-Bi |
Fig. 2 CO2-utilization bypass model diagram of simbiology
Part C : (Original metabolism model)
It’s a original pathway which xylose is consumed by E.coli. Basically, E.coli will comsume the xylose and then produce the temporary production, pyruvate.
| Equation | Enzyme | Kinetic law |
|---|---|---|
| $${X5P + R5P \rightarrow S7P + GAP}$$ | transketolase | Henri-Michaelis-Menten |
| $${S7P + GAP \rightarrow F6P + E4P}$$ | transaldolase | Henri-Michaelis-Menten |
| $${E4P + X5P \rightarrow F6P + GAP}$$ | transaldolase | Henri-Michaelis-Menten |
| $${FBP \rightarrow GAP}$$ | fructose-bisphosphate aldolase | Henri-Michaelis-Menten |
| $${DHAP \rightarrow GAP}$$ | triose-phosphate isomerase | Reversible Henri-Michaelis-Menten |
| $${F6P + ATP \rightarrow FBP + ADP}$$ | 6-phosphofructokinase | Henri-Michaelis-Menten |
| $${F6P \rightarrow GAP}$$ | glucose-6-phosphate isomerase | Henri-Michaelis-Menten |
| $${G6P + NADP^+ \rightarrow 6PG + NAPDH}$$ | 6-phosphogluconolactonase | Ping-Pong-Bi-Bi |
| $${6PG + NADP^+ \rightarrow Ru5P + NAPDH}$$ | phosphogluconate dehydrogenase (NADP+-dependent, decarboxylating) | Henri-Michaelis-Menten |
| $${GAP + NADP^+ + ADP \rightarrow 3PGA + NAPDH + ATP}$$ | glyceraldehyde-3-phosphate dehydrogenase (NADP+) | Henri-Michaelis-Menten |
| phosphoglycerate kinase | ||
| $${3PGA \rightarrow PEP}$$ | phosphoglycerate mutase | Henri-Michaelis-Menten |
| phosphopyruvate hydratase | ||
| $${PEP \rightarrow PYR}$$ | pyruvate kinase | Henri-Michaelis-Menten |
Fig. 3 Original metabolism model diagram of simbiology
Part D : (TCA cycle model)
In Tricarboxylic acid cycle (TCA cycle), we can get final production in this cycle. The two carbons of Acetyl CoA are decomposed into carbon dioxide to produce energy, form ATP or GTP, and reduce NADH and FADH2, and then provide the energy needed for the reaction in the organism.
| Equation | Enzyme | Kinetic law |
|---|---|---|
| $${PEP + HCO_3^- + NAD^+ \rightarrow OAA + NADH}$$ | PEP carboxylase | Reversible Henri-Michaelis-Menten |
| $${AcCoA \rightarrow CIT}$$ | citrate synthase | Reversible Henri-Michaelis-Menten |
| $${cIT \rightarrow ICIT}$$ | aconitate hydratase | Reversible Henri-Michaelis-Menten |
| $${ICIT + NADP^+ \rightarrow AKG + NADPH + CO_2}$$ | isocitrate dehydrogenase | Reversible Henri-Michaelis-Menten |
| $${AKG + NAD^+ \rightarrow SUCCoA + NADH + CO_2}$$ | dihydrolipoyllysine-residue succinyltransferase | Reversible Henri-Michaelis-Menten |
| $${SUCCoA + ADP \rightarrow SUCC + ATP}$$ | succinate-CoA ligase (ADP-forming) | Reversible Henri-Michaelis-Menten |
| $${SUCC + FAD \rightarrow FUM + FADH_2}$$ | succinate dehydrogenase | Reversible Henri-Michaelis-Menten |
| $${FUM \rightarrow MAL}$$ | fumarate hydratase | Reversible Henri-Michaelis-Menten |
| $${MAL + NAD^+ \rightarrow OAA + NADH}$$ | malate dehydrogenase | Reversible Henri-Michaelis-Menten |
| $${OAA \rightarrow CIT}$$ | citrate synthase | Reversible Henri-Michaelis-Menten |
| $${PYR \rightarrow NADH + AcCoA + CO_2}$$ | pyruvate dehydrogenase complex | Henri-Michaelis-Menten |
| $${ICIT \rightarrow GOX}$$ | isocitrate lyase | Reversible Henri-Michaelis-Menten |
| $${GOX \rightarrow MAL}$$ | malate synthase | Reversible Henri-Michaelis-Menten |
| $${ICIT \rightarrow SUCC + GOX}$$ | isocitrate lyase | Reversible Henri-Michaelis-Menten |
Fig. 4 TCA cycle model diagram of simbiology
Result analysis
We built the whole xylose metabolism in our engineered E. coli, which help us calculate the rate of xylose will go into CO2 utilization bypass. The result of this model shows that CO2 uptake affect the production of pyruvate. We can confirm that our engineered E. coli successfully uptake CO2 while the increase rate of pyruvate production followed the experiment data. To see more analysis of CO2 utilization on analysis page.
Fig. 5 TCA cycle model diagram of simbiology
Fig. 6 whole xylose metabolisml diagram of simbiology
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