Difference between revisions of "Team:NCKU Tainan/Model"
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<a class="list-group-item list-group-item-action" href="#achievement">Achievement</a> | <a class="list-group-item list-group-item-action" href="#achievement">Achievement</a> | ||
<a class="list-group-item list-group-item-action" href="#purpose">Purpose</a> | <a class="list-group-item list-group-item-action" href="#purpose">Purpose</a> | ||
| + | <a class="list-group-item list-group-item-action" href="#method">Method</a> | ||
<a class="list-group-item list-group-item-action" href="#CO2_uptake">CO<sub>2</sub> uptake</a> | <a class="list-group-item list-group-item-action" href="#CO2_uptake">CO<sub>2</sub> uptake</a> | ||
<a class="list-group-item list-group-item-action" href="#CO2_metabolism">CO<sub>2</sub> metabolism</a> | <a class="list-group-item list-group-item-action" href="#CO2_metabolism">CO<sub>2</sub> metabolism</a> | ||
| Line 56: | Line 57: | ||
<a class="link" href="#CO2_uptake">CO<sub>2</sub> uptake</a> and <a class="link" href="#CO2_metabolism">CO<sub>2</sub> metabolism</a>. | <a class="link" href="#CO2_uptake">CO<sub>2</sub> uptake</a> and <a class="link" href="#CO2_metabolism">CO<sub>2</sub> metabolism</a>. | ||
</p> | </p> | ||
| + | </div> | ||
| + | <div id="method"> | ||
| + | <h3>Method of model</h3> | ||
| + | <p class="pcontent">Follow our model progress diagram with a better sense of how we achieve our goal.</p> | ||
| + | <div class="row"> | ||
| + | <div class="col-3"></div> | ||
| + | <div class="col-6" id="cube1"> | ||
| + | <h4 class="pb">Purpose of model</h4> | ||
| + | <p class="pb">How many CO<sub>2</sub> can be used by <i>E. coli</i></p> | ||
| + | </div> | ||
| + | <div class="col-3"></div> | ||
| + | </div> | ||
| + | <div class="row"> | ||
| + | <div class="col-12"> | ||
| + | <p class="pcontent" id="arrowdown"><i class="fa fa-arrow-down"></i></p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row"> | ||
| + | <div class="col-3"></div> | ||
| + | <div class="col-6" id="cube2"> | ||
| + | <a class="methodlink" href="https://2018.igem.org/Team:NCKU_Tainan/Kinetic_Law"> | ||
| + | <h4 class="pb">Method of model</h4> | ||
| + | <p class="pb">Kinetic law / Metabolic Pathway / Chemical Equation</p> | ||
| + | </a> | ||
| + | </div> | ||
| + | <div class="col-3"></div> | ||
| + | </div> | ||
| + | <div class="row"> | ||
| + | <div class="col-12"> | ||
| + | <p class="pcontent" id="arrowdown"><i class="fa fa-arrow-down"></i></p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row"> | ||
| + | <div class="col-3"></div> | ||
| + | <div class="col-6" id="cube1"> | ||
| + | <a class="methodlink" href="https://2018.igem.org/Team:NCKU_Tainan/Kinetic_Law"> | ||
| + | <h4 class="pb">Collect experiment data</h4> | ||
| + | </a> | ||
| + | </div> | ||
| + | <div class="col-3"></div> | ||
| + | </div> | ||
| + | <div class="row"> | ||
| + | <div class="col-12"> | ||
| + | <p class="pcontent" id="arrowdown"><i class="fa fa-arrow-down"></i></p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row"> | ||
| + | <div class="col-3"></div> | ||
| + | <div class="col-6" id="cube3"> | ||
| + | <a class="methodlink" href="https://2018.igem.org/Team:NCKU_Tainan/Analyse"> | ||
| + | <h4 class="pb">Result analysis</h4> | ||
| + | </a> | ||
| + | </div> | ||
| + | <div class="col-3"></div> | ||
| + | </div> | ||
</div> | </div> | ||
<div id="CO2_uptake"> | <div id="CO2_uptake"> | ||
| Line 122: | Line 178: | ||
<div class="row"> | <div class="row"> | ||
<h4>Result</h4> | <h4>Result</h4> | ||
| + | <p class="pcontent">Enzyme, CA, strongly catalyzes the hydration of CO<sub>2</sub> and its interaction appears to | ||
| + | follow a Michaelis-Menten mechanism with a Hill constant of 0.0062 in our research. | ||
| + | The result of Engineered <i>E. coli</i> cloned with or without CA gene showed below. | ||
| + | A quite different diffusion time explains the function of CA. | ||
| + | </p> | ||
<div class="col-12" id="resultimg"> | <div class="col-12" id="resultimg"> | ||
<img class="twoimg" src="https://static.igem.org/mediawiki/2018/7/75/T--NCKU_Tainan--model_result1.png"> | <img class="twoimg" src="https://static.igem.org/mediawiki/2018/7/75/T--NCKU_Tainan--model_result1.png"> | ||
| Line 695: | Line 756: | ||
<li class="smallp">Jacqueline E. G, Christopher P. L, Maciek R. A. Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by 13C metabolic flux analysis. Metab Eng. 2017 Jan; 39: 9–18.</li> | <li class="smallp">Jacqueline E. G, Christopher P. L, Maciek R. A. Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by 13C metabolic flux analysis. Metab Eng. 2017 Jan; 39: 9–18.</li> | ||
<li class="smallp">Uwe Sauer, Bernhard J. E. The PEP—pyruvate—oxaloacetate node as the switch point for carbon flux distribution in bacteria. FEMS Microbiology Reviews, Volume 29, Issue 4, 1 September 2005, Pages 765–794.</li> | <li class="smallp">Uwe Sauer, Bernhard J. E. The PEP—pyruvate—oxaloacetate node as the switch point for carbon flux distribution in bacteria. FEMS Microbiology Reviews, Volume 29, Issue 4, 1 September 2005, Pages 765–794.</li> | ||
| + | <li class="smallp">Fuyu G, Guoxia L, Xiaoyun Z, Jie Z, Zhen C and Yin L. Quantitative analysis of an engineered CO2-fixing Escherichia coli reveals great potential of heterotrophic CO2 fixation. Gong et al. Biotechnology for Biofuels, 2015, 8:86.</li> | ||
| + | <li class="smallp">Y. Pocker, and Joan S. Y. Ng. Plant carbonic anhydrase. Properties and carbon dioxide hydration kinetics. Biochemistry, 1973, 12 (25), pp 5127–5134.</li> | ||
</ol> | </ol> | ||
<br class="pcontent"> | <br class="pcontent"> | ||
Revision as of 17:14, 22 September 2018
Model
Achievement
- Figure out how much CO2 uptake into the E.coli to make our project more effective.
- We will realize how much carbon we can fix in our project.
In order to achieve these two goals, we try to figure out three main questions：
Purpose
We aim to develop the biological model to make our experimental data more useful, even more effective to achieve the optimization parameters in the model. Therefore, we can save time to try and error on doing experiment. After that, we can analyze the rate of production and consumption. In this way, we can calculate the amount of carbon dioxide uptake into the Escherichia coli (E.coli) and calculate how many CO2 can be used in our system. In addition, we also want to realize how much carbon we can fix. We have to understand what’s going on in E. coli after uptaking CO2. Since we integrated non-native carbon fixation pathway into E. coli to let E. coli utilize CO2, we can simplify the CO2 utilization pathway in engineered E. coli into two parts, CO2 uptake and CO2 metabolism.
Method of model
Follow our model progress diagram with a better sense of how we achieve our goal.
Purpose of model
How many CO2 can be used by E. coli
CO2 uptake
CO2 exists in the phase of gas. Therefore, E. coli uptake CO2 by diffusion. However, the native diffusion rate is really low, not to mention the dissolved rate. We clone CA (carbonic anhydrase) gene to speed up the diffusion rate. The higher diffusion rate, the higher CO2 concentration maintained in engineered E. coli. Higher CO2 concentration in engineered E. coli provides more steady CO2 condition for downstream pathway.
| CO2 uptake | Kf | Kr | |||||
|---|---|---|---|---|---|---|---|
| R1 | $${[CO_2]_{air} \rightarrow [CO_2]_{uptake}}$$ | 9.23E-04 | 4.78E-04 | ||||
| R2 | $${[CO_2]_{uptake} + [H_2O] \rightarrow [H_2CO_3]}$$ | 0.062 | 23.7 | ||||
| R3 | $${[H_2CO_3] \rightarrow [H^+] + [HCO_3^-]}$$ | 8000000 | 4.7E10 | ||||
| R4 | $${[HCO_3^-] \rightarrow [H^+] + [CO_3^{2-}]}$$ | 3 | 5E10 | ||||
| R5 | $${[H^+] + [HCO_3^-] \longleftrightarrow [H_2O] + [CO_2]_{uptake}}$$ | Vmax | Km | ||||
| 0.47333 | 0.0062 | ||||||
Result
Enzyme, CA, strongly catalyzes the hydration of CO2 and its interaction appears to follow a Michaelis-Menten mechanism with a Hill constant of 0.0062 in our research. The result of Engineered E. coli cloned with or without CA gene showed below. A quite different diffusion time explains the function of CA.
Fig. 2 CO2 uptake amount change with time in engineered E. coli without CA gene.
Fig. 3 CO2 uptake amount change with time in engineered E. coli with CA gene.
CO2 Metabolism
Xylose is the main source of our engineered E. coli to uptake CO2. However, there is another pathway for xylose metabolism, which means that there is only a part of xylose that E. coli consumed will react with CO2. We build a model to predict the different carbon flux between original xylose metabolism, PP pathway (Pentose Phosphate pathway), and our CO2 bypass pathway.
Both of two pathways will end up at pyruvate, which is a key intersection in the network of metabolic pathways. We built a model of TCA cycle to explain how pyruvate play an energy precursor role and then is converted into biomass of E. coli.
Pathway
Fig. 4 Metabolic pathway of CO2-utilization E. coli. The introduced CO2-utilization bypass pathway composed of PRK and Rubisco is drawn in green line and noted by ” A” reaction, while the central carbon metabolic pathway including PP pathway and TCA cycle is drawn in blue line and yellow line and noted by “B” reaction and “C” reaction, respectively. See more detail in Kinetic law.
| CO2 bypass pathway | Vmax (1/S) | KM (mM) | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A1 | $${Xylose \rightarrow Xylulose}$$ $${Xylulose + ATP \rightarrow X5P}$$ | 0.02456 | 124.55879 | ||||||||||
| A2 | $${X5P \rightarrow Ru5P}$$ | 0.08 | 12.5 | ||||||||||
| A3 | $${Ru5P \xrightarrow{PRK} RuBP}$$ | 225.81858 | 0.27 | ||||||||||
| A4 | $${RuBP + CO_2 \xrightarrow{RuBisCO} 3PG}$$ | Vmax | Krubp | KCO2 | |||||||||
| 333.33 | 53 | 2.7 | |||||||||||
| Pentose Phosphate Pathway | Vmax (1/S) | KM (mM) | ||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| B1 | $${Ru5P \rightarrow R5P}$$ | 0.13672 | 12.5 | |||||||||||||||||||||
| B2 | $${X5P + R5P \rightarrow S7P + GAP}$$ | |||||||||||||||||||||||
| $${X5P \rightarrow S7P}$$ | 58.27 | 4.96E-09 | ||||||||||||||||||||||
| $${X5P \rightarrow GAP}$$ | 58.27 | 48 | ||||||||||||||||||||||
| $${R5P \rightarrow S7P}$$ | 58.27 | 0.008 | ||||||||||||||||||||||
| $${R5P \rightarrow GAP}$$ | 58.27 | 3.05E-07 | ||||||||||||||||||||||
| B3 | $${S7P + GAP \rightarrow F6P + E4P}$$ | |||||||||||||||||||||||
| $${S7P \rightarrow E4P}$$ | 58.27 | 2.524 | ||||||||||||||||||||||
| $${S7P \rightarrow F6P}$$ | 58.27 | 6.10E-09 | ||||||||||||||||||||||
| $${GAP \rightarrow E4P}$$ | 58.27 | 1.73E-07 | ||||||||||||||||||||||
| $${GAP \rightarrow F6P}$$ | 58.27 | 8.23E-09 | ||||||||||||||||||||||
| B4 | $${E4P + X5P \rightarrow F6P + GAP}$$ | |||||||||||||||||||||||
| $${E4P \rightarrow F6P}$$ | 1 | 1 | ||||||||||||||||||||||
| $${E4P \rightarrow GAP}$$ | 5.827E-4 | 1.733E-7 | ||||||||||||||||||||||
| $${X5P \rightarrow F6P}$$ | 0.05827 | 1.733E-7 | ||||||||||||||||||||||
| $${X5P \rightarrow GAP}$$ | 0.005827 | 48.8 | ||||||||||||||||||||||
| B5 | $${FBP \rightarrow GAP}$$ | 135.425 | 4 | |||||||||||||||||||||
| B6 | $${DHAP \longleftrightarrow GAP}$$ | Vmf | Vmr | KmDHAP | KmGAP | |||||||||||||||||||
| 0.2394 | 0.24 | 0.812 | 0.245 | |||||||||||||||||||||
| B7 | $${FBP \rightarrow DHAP}$$ | 0.0001247 | 0.04 | |||||||||||||||||||||
| B8 | $${F6P + ATP \rightarrow FBP + ADP}$$ | 88.2533 | 0.017 | |||||||||||||||||||||
| B9 | $${F6P \rightarrow G6P}$$ | 88.2353 | 0.017 | |||||||||||||||||||||
| B10 | $${G6P + NADP^+ \rightarrow 6PG + NADPH}$$ | Vmax | KmG6P | KmNADP | ||||||||||||||||||||
| 11.736 | 0.12 | 0.0123 | ||||||||||||||||||||||
| B11 | $${6PG + NADP^+ \rightarrow Ru5P + NADPH}$$ | 1.0 | 0.044 | |||||||||||||||||||||
| B12 | $${GAP + NADP^+ + ADP \rightarrow 3PGA + NADPH + ATP}$$ | 0.4 | 0.013 | |||||||||||||||||||||
| B13 | $${3PGA \rightarrow PEP}$$ | 0.43478 | 0.23 | |||||||||||||||||||||
| B14 | $${PEP \rightarrow PYR}$$ | 0.23148 | 0.45 | |||||||||||||||||||||
| TCA cycle | Vmf (mM/min) | Vmr (mM/min) | Ks (mM) | Kp (mM) | ||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| C1 | $${PEP + HCO_3^- + NAD^+ \rightarrow OAA + NADH}$$ | 1 | 1 | 1 | 1 | |||||||||||||||||||
| C2 | $${AcCoA \rightarrow CIT}$$ | 64.8 | 0.648 | 0.05 | 0.12 | |||||||||||||||||||
| C3 | $${CIT \rightarrow ICIT}$$ | 31.2 | 0.312 | 1.7 | 0.7 | |||||||||||||||||||
| C4 | $${ICIT + NADP^+ \rightarrow AKG + NADPH + CO_2}$$ | 10.2 | 0.102 | 0.03 | 0.3 | |||||||||||||||||||
| C5 | $${AKG + NAD^+ \rightarrow AKG + NADPH + CO_2}$$ | 57.344 | 0.57344 | 0.1 | 1.0 | |||||||||||||||||||
| C6 | $${SUCCoA + ADP \rightarrow SUCC + ATP}$$ | 6.54 | 0.0651 | 0.015 | 0.15 | |||||||||||||||||||
| C7 | $${SUCC + FAD \rightarrow FUM + FADH_2}$$ | 1.02 | 1.02 | 0.12 | 0.15 | |||||||||||||||||||
| C8 | $${FUM \rightarrow MAL}$$ | 887.7 | 87.7 | 0.25 | 2.38 | |||||||||||||||||||
| C9 | $${MAL + NAD^+ \rightarrow OAA + NADH}$$ | 184 | 184 | 0.833 | 0.0443 | |||||||||||||||||||
| C10 | $${OAA \rightarrow CIT}$$ | 64.8 | 0.648 | 0.012 | 0.12 | |||||||||||||||||||
| C11 | $${PYR \rightarrow NADH + AcCoA + CO_2}$$ | Vmax (1/s) | KM (mM) | |||||||||||||||||||||
| 0.00518 | 1.5 | |||||||||||||||||||||||
| C12 | $${ICIT \rightarrow GOX}$$ | 1.172 | 0.01172 | 0.145 | 0.13 | |||||||||||||||||||
| C13 | $${GOX \rightarrow MAL}$$ | 20 | 0.2 | 0.057 | 1.0 | |||||||||||||||||||
| C14 | $${ICIT \rightarrow SUCC + GOX}$$ | Vmf (mM/min) | Vmr (mM/min) | Kicit (mM) | Ksucc (mM) | Kgox (mM) | ||||||||||||||||||
| 1.172 | 0.012 | 0.145 | 0.59 | 0.13 | ||||||||||||||||||||
| E4P | erythrose-4-phosphate |
| F6P | fructose-6-phosphate |
| FBP | fructose-1,6-bisphosphate |
| G6P | glucose-6-phosphate |
| GAP | glyceraldehyde-3-phosphate |
| PEP | phosphoenol pyruvate |
| PYR | pyruvate |
| R5P | ribose-5-phosphate |
| Ru5P | ribulose-5-phosphate |
| S7P | sedoheptulose-7-phosphate |
| X5P | xylulose-5-phosphate |
| 6PG | 6-phosphogluconate |
| DHAP | Dihydroxyacetone phosphate |
| 3PG | 3-Phosphoglyceric acid |
| RuBP | ribulose-1,5-bisphosphate |
| PRK | Phosphoribulokinase |
| Rubisco | Ribulose-1,5-bisphosphate carboxylase/ oxygenase |
| AcCoA | Acetyl-CoA |
| CIT | Citrate |
| ICIT | Isocitrate |
| AKG | Alpha-ketoglutarate |
| SUCCoA | Succinyl-CoA |
| SUCC | Succinate |
| FUM | Fumarate |
| MAL | Malate |
| OAA | Oxaloacetic acid |
| GOX | Glyoxylate |
Xylose v.s. CO2 analysis in CO2 utilization efficiency
Two main sources of CO2 metabolism in engineered E. coli are xylose and CO2. CO2 utilization rate varies under different condition. We use 5% CO2 (about 2.6 mM) and 0.4% (about 26 mM) xylose in experiment as an optimization CO2 utilization rate. Constant CO2 condition and limited CO2 condition also effects metabolism performance. In other words, open system and close system showed different results. Therefore, adjust initial concentration of CO2 and Xylose and choose open or close system to see the change of pyruvate production.
Reference
- Jacqueline E. G, Christopher P. L, Maciek R. A. Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by 13C metabolic flux analysis. Metab Eng. 2017 Jan; 39: 9–18.
- Uwe Sauer, Bernhard J. E. The PEP—pyruvate—oxaloacetate node as the switch point for carbon flux distribution in bacteria. FEMS Microbiology Reviews, Volume 29, Issue 4, 1 September 2005, Pages 765–794.
- Fuyu G, Guoxia L, Xiaoyun Z, Jie Z, Zhen C and Yin L. Quantitative analysis of an engineered CO2-fixing Escherichia coli reveals great potential of heterotrophic CO2 fixation. Gong et al. Biotechnology for Biofuels, 2015, 8:86.
- Y. Pocker, and Joan S. Y. Ng. Plant carbonic anhydrase. Properties and carbon dioxide hydration kinetics. Biochemistry, 1973, 12 (25), pp 5127–5134.