Team:Aberdeen Scotland/Equations
From 2010.igem.org
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<h1>Equations</h1> | <h1>Equations</h1> | ||
- | <p>Here we define the equations and parameters that describe the novel genetic toggle switch that works at the translational level. The switch allows mutually exclusive expression of either green fluorescent protein (GFP) or cyan | + | <p>Here we define the equations and parameters that describe the novel genetic toggle switch that works at the translational level. The switch allows mutually exclusive expression of either green fluorescent protein (GFP) or cyan fluorescent protein (CFP). The synthetic biological circuit is represented in Fig 1.</p> |
- | fluorescent protein (CFP). The synthetic biological circuit is represented in Fig | + | |
<br> | <br> | ||
<center> | <center> | ||
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<br> | <br> | ||
- | <p>We can regulate the system when we add galactose or | + | <p>We can regulate the system when we add galactose or methionine. Galactose will bind to the GAL promoter and activate the transcription of M1, allowing the system to express GFP. If we remove methionine from the system instead of adding galactose, it will bind to the MET1 promoter, the transcription of M2 will be activated, leading to the expression of CFP.</p> |
- | to the | + | |
<br> | <br> | ||
<p>From Fig 1 it can be seen that there is mutual inhibition of the translation of the two mRNAs. That is because the translated proteins can bind to the corresponding stem loop structures on the opposing construct.</p> | <p>From Fig 1 it can be seen that there is mutual inhibition of the translation of the two mRNAs. That is because the translated proteins can bind to the corresponding stem loop structures on the opposing construct.</p> | ||
<br> | <br> | ||
- | <p>For our initial conditions, we began with more GFP than CFP and thus the production of CFP was inhibited. When | + | <p>For our initial conditions, we began with more GFP than CFP and thus the production of CFP was inhibited. When methionine was added removed from the system, the rate of CFP production will increase and decrease for GFP. Eventually, we will see more CFP than GFP so the system will have switched. Once we have more CFP than GFP, galactose can then be added to switch back to an expression of GFP. </p> |
<br> | <br> | ||
<p>The N-Peptide and GFP strand has two MS2-Stem loops as we discovered that one single loop would not inhibit the production of CFP enough to achieve our switch.</p> | <p>The N-Peptide and GFP strand has two MS2-Stem loops as we discovered that one single loop would not inhibit the production of CFP enough to achieve our switch.</p> | ||
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<br> | <br> | ||
<h3>Equation 1</h3> | <h3>Equation 1</h3> | ||
- | + | <br> | |
<div align="center"> | <div align="center"> | ||
<table> | <table> | ||
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</table> | </table> | ||
</div> | </div> | ||
- | + | <br> | |
+ | <p>This is the equation for the rate of change of the mRNA that is transcribed from the galactose promoter. The three terms represent production, degradation, and dilution respectively.</p> | ||
+ | <br> | ||
+ | <p>[GAL] represents the concentration of galactose that is added to the system. When galactose is added it binds to the promoter and activates the transcription of M1. | ||
+ | <br> | ||
+ | <br> | ||
+ | [M1] is the concentration of mRNA that translates the N-peptide and GFP.</p> | ||
+ | <br> | ||
<div align="center"> | <div align="center"> | ||
<table> | <table> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | <div align="right"><b><p> | + | <div align="right"><b><p>Parameter<p></b></div> |
</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p><b>Description</b></p> |
</td> | </td> | ||
</tr> | </tr> | ||
- | + | <tr> | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
<td> | <td> | ||
<div align="right"><b><p>λ<sub style="font-size:10px">1</sub>:<p></b></div> | <div align="right"><b><p>λ<sub style="font-size:10px">1</sub>:<p></b></div> | ||
</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p>Constant representing rate of transcription of the DNA that encodes for the production of N peptide and GFP</p> |
</td> | </td> | ||
</tr> | </tr> | ||
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</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p>Constant representing rate of degradation of mRNA</p> |
</td> | </td> | ||
</tr> | </tr> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
- | <div align="right"><b><p> | + | <div align="right"><b><p>K<sub style="font-size:10px">1</sub>:</p></b></div> |
</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p>Dissociation constant for the GAL promoter</p> |
</td> | </td> | ||
</tr> | </tr> | ||
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</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p>Time constant representing rate of cellular division</p> |
</td> | </td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
+ | <br> | ||
+ | <h3>Equation 2</h3> | ||
<br> | <br> | ||
- | |||
- | |||
- | |||
- | |||
<div align="center"> | <div align="center"> | ||
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</table> | </table> | ||
</div> | </div> | ||
- | + | <br> | |
+ | <p>This is the equation for the rate of change of protein that is translated from the mRNA for GFP. The three terms represent production, degradation, and dilution respectively.</p> | ||
+ | <br> | ||
+ | <p>[M1] is the concentration of mRNA that translates the N-peptide GFP. | ||
+ | <br> | ||
+ | <br> | ||
+ | [GFP] represents the concentration of N-peptide and GFP. | ||
+ | <br> | ||
+ | <br> | ||
+ | [CFP] represents the concentration of the MS2-protein and CFP.</p> | ||
+ | <br> | ||
<div align="center"> | <div align="center"> | ||
<table> | <table> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | <div align="right"><b><p> | + | <div align="right"><b><p>Parameter:<p></b></div> |
</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p><b>Description</b></p> |
</td> | </td> | ||
</tr> | </tr> | ||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
<tr> | <tr> | ||
<td> | <td> | ||
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</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p>Constant representing rate of translation of the mRNA that encodes for the production of N-peptide and GFP</p> |
</td> | </td> | ||
</tr> | </tr> | ||
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</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p>Constant representing rate of degradation of the GFP</p> |
</td> | </td> | ||
</tr> | </tr> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
- | <div align="right"><b><p> | + | <div align="right"><b><p>K<sub style="font-size:10px">2</sub>:</p></b></div> |
</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p>Dissociation constant for the MS2-CFP protein to MS2 loop</p> |
</td> | </td> | ||
</tr> | </tr> | ||
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</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p>Time constant representing rate of cellular division</p> |
</td> | </td> | ||
</tr> | </tr> | ||
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<br> | <br> | ||
+ | <h3>Equation 3</h3> | ||
<br> | <br> | ||
- | |||
- | |||
- | |||
<div align="center"> | <div align="center"> | ||
<table> | <table> | ||
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</table> | </table> | ||
</div> | </div> | ||
+ | <br> | ||
+ | <p>This is the equation for the rate of change of the mRNA that is transcribed from the copper promoter. The three terms represent production, degradation, and dilution respectively.</p> | ||
+ | <br> | ||
+ | <p>[Cu<sup style="font-size:10px">2+</sup>] is the concentration of the copper added to the system that binds to the CUP1 promoter and activates the transcription of M2. | ||
+ | <br> | ||
+ | <br> | ||
+ | [M2] represents the concentration of mRNA that translates the MS2-protein and CFP. </p> | ||
+ | <br> | ||
<div align="center"> | <div align="center"> | ||
<table> | <table> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | <div align="right"><b><p> | + | <div align="right"><b><p>Parameter</p></b></div> |
</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p><b>Description</b></p> |
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | <div align="right"><b><p> | + | <div align="right"><b><p>λ<sub style="font-size:10px">3</sup>:<p></b></div> |
</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p>Constant representing rate of transcription of the DNA that encodes for the production of the MS2-protein and CFP</p> |
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | <div align="right"><b><p> | + | <div align="right"><b><p>μ<sub style="font-size:10px">3</sub>:</p></b></div> |
</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p>Constant representing rate of degradation of mRNA</p> |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | <div align="right"><b><p> | + | <div align="right"><b><p>n<sub style="font-size:10px">3</sub>:<p></b></div> |
</td> | </td> | ||
<td> | <td> | ||
- | <p>Hill coefficient<p> | + | <p>Hill coefficient of the association between copper and the CUP1 promoter<p> |
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | <div align="right"><b><p> | + | <div align="right"><b><p>K<sub style="font-size:10px">3</sub>:</p></b></div> |
</td> | </td> | ||
<td> | <td> | ||
- | <p>constant | + | <p>Dissociation constant for Copper promoter</p> |
</td> | </td> | ||
</tr> | </tr> | ||
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</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p>Time constant representing rate of cellular division</p> |
</td> | </td> | ||
</tr> | </tr> | ||
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<br> | <br> | ||
+ | |||
+ | <h3>Equation 4</h3> | ||
<br> | <br> | ||
- | |||
- | |||
- | |||
- | |||
- | |||
<div align="center"> | <div align="center"> | ||
<table> | <table> | ||
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</table> | </table> | ||
</div> | </div> | ||
+ | <br> | ||
+ | <p>This is the equation for the rate of change of protein that is translated from the mRNA for CFP. The three terms represent production, degradation, and dilution respectively.</p> | ||
+ | <br> | ||
+ | <p>[M2] is the concentration of mRNA that translates to MS2-protein and CFP. | ||
+ | <br> | ||
+ | <br> | ||
+ | [GFP] represents the concentration of the N-peptide and GFP. | ||
+ | <br> | ||
+ | <br> | ||
+ | [CFP] represents the concentration of the MS2-protein and CFP.</p> | ||
+ | <br> | ||
<div align="center"> | <div align="center"> | ||
<table> | <table> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | <div align="right"><b><p> | + | <div align="right"><b><p>Parameters<p></b></div> |
</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p><b>Description</b></p> |
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | <div align="right"><b><p> | + | <div align="right"><b><p>λ<sub style="font-size:10px">4</sub>:</p></b></div> |
</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p>Constant representing rate of translation of the mRNA that encodes for the production of MS2-protein and CFP</p> |
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | <div align="right"><b><p> | + | <div align="right"><b><p>μ<sub style="font-size:10px">4</sub>:</p></b></div> |
</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p>Constant representing rate of degradation of the CFP</p> |
</td> | </td> | ||
+ | </tr> | ||
<tr> | <tr> | ||
+ | <td> | ||
+ | <div align="right"><b><p>n<sub style="font-size:10px">4</sub>:<p></b></div> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p>Hill coefficient of the GFP/Bbox stem loop association<p> | ||
+ | </td> | ||
+ | </tr> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | <div align="right"><b><p> | + | <div align="right"><b><p>K<sub style="font-size:10px">4</sub>:</p></b></div> |
</td> | </td> | ||
<td> | <td> | ||
- | <p>constant | + | <p>Dissociation constant for the N-Pep-GFP protein to the Bbox-stem loop</p> |
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | <div align="right"><b><p> | + | <div align="right"><b><p>T:</p></b></div> |
</td> | </td> | ||
<td> | <td> | ||
- | <p>constant representing | + | <p>time constant representing rate of cellular division</p> |
</td> | </td> | ||
</tr> | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <br> | ||
+ | <h1>Parameter Study</h1> | ||
+ | <p>The parameter values were first estimated based on the literature <a href="#ref1"><sup style="font-size:10px">[1]</sup></a> and after the first estimation, a possible range of variation for each parameter was assigned, also based on literature. Then, we studied the bistability of the model depending on the parameter values that were varied in the above mentioned ranges. For more information, see <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Probability">Parameter Space Analysis</a> and <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Evolution">Directed Evolution</a>.</p> | ||
+ | <br> | ||
+ | <h1>Modification of the construct</h1> | ||
+ | |||
+ | <p>Some experimental difficulties were encountered with the copper construct which led to the use of a methionine promoter to substitute it. Methionine acts as an inhibitor of the promoter, so that equation 3 had to be substituted by the following equation:<p> | ||
+ | <br> | ||
+ | <center> | ||
+ | <img src="https://static.igem.org/mediawiki/2010/f/f2/Meth.png"> | ||
+ | <br><br> | ||
+ | <img src="https://static.igem.org/mediawiki/2010/7/75/MET_toggle_switch.png"> | ||
+ | </center> | ||
+ | <br> | ||
+ | <p>The behaviour of the switch can then be summarise in the following table:</p> | ||
+ | <br> | ||
+ | <div align="center"> | ||
+ | <table> | ||
<tr> | <tr> | ||
- | <td> | + | <td> |
- | <div align="right"><b><p> | + | <div align="right"><b><p>What is present in the system</p></b></div> |
</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p><b>Protein(s) produced</b></p> |
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | <div align="right" | + | <div align="right"><p>Galactose and Methionine</p></div> |
</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p>GFP</p> |
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> | <td> | ||
- | <div align="right" | + | <div align="right"><p>Galactose only</p></div> |
</td> | </td> | ||
<td> | <td> | ||
- | <p> | + | <p>GFP, CFP (doses dependent)</p> |
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <div align="right"><p>Methionine only</p></div> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p>No GFP or CFP</p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <div align="right"><p>No Galactose and no Methionine</p></div> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p>CFP</p> | ||
</td> | </td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
+ | <br> | ||
+ | |||
+ | <h1>References</h1> | ||
+ | <a name="ref1"></a> | ||
+ | <p><sup style="font-size:10px">[1]</sup> Beyer A, Hollunder J, Nasheuer HP, Wilhelm T. (2004), Post-transcriptional expression regulation in the yeast Saccharomyces cerevisiae on a genomic scale, <i>Mol Cell Proteomics.</i>, Vol. 3, No.11, pp. 1083-1092.</p> | ||
+ | <br> | ||
+ | <p><sup style="font-size:10px">[2]</sup> Alon, U. (2006), An Introduction to Systems Biology: Design Principles of Biological Circuits, Chapman and Hall. </p> | ||
+ | |||
+ | |||
<br><br> | <br><br> | ||
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</tr> | </tr> | ||
</table> | </table> | ||
+ | |||
+ | |||
</html> | </html> | ||
+ | {{:Team:Aberdeen_Scotland/Footer}} |
Latest revision as of 20:29, 27 October 2010
University of Aberdeen - ayeSwitch
Equations
Here we define the equations and parameters that describe the novel genetic toggle switch that works at the translational level. The switch allows mutually exclusive expression of either green fluorescent protein (GFP) or cyan fluorescent protein (CFP). The synthetic biological circuit is represented in Fig 1.
![](https://static.igem.org/mediawiki/2010/f/ff/Toggle_switch.jpg)
Figure 1: Translation of DNA to mRNA.
We can regulate the system when we add galactose or methionine. Galactose will bind to the GAL promoter and activate the transcription of M1, allowing the system to express GFP. If we remove methionine from the system instead of adding galactose, it will bind to the MET1 promoter, the transcription of M2 will be activated, leading to the expression of CFP.
From Fig 1 it can be seen that there is mutual inhibition of the translation of the two mRNAs. That is because the translated proteins can bind to the corresponding stem loop structures on the opposing construct.
For our initial conditions, we began with more GFP than CFP and thus the production of CFP was inhibited. When methionine was added removed from the system, the rate of CFP production will increase and decrease for GFP. Eventually, we will see more CFP than GFP so the system will have switched. Once we have more CFP than GFP, galactose can then be added to switch back to an expression of GFP.
The N-Peptide and GFP strand has two MS2-Stem loops as we discovered that one single loop would not inhibit the production of CFP enough to achieve our switch.
Equation 1
![]() |
(1) |
This is the equation for the rate of change of the mRNA that is transcribed from the galactose promoter. The three terms represent production, degradation, and dilution respectively.
[GAL] represents the concentration of galactose that is added to the system. When galactose is added it binds to the promoter and activates the transcription of M1.
[M1] is the concentration of mRNA that translates the N-peptide and GFP.
Parameter |
Description |
λ1: |
Constant representing rate of transcription of the DNA that encodes for the production of N peptide and GFP |
μ1: |
Constant representing rate of degradation of mRNA |
n1: |
Hill coefficient for the association between the galactose and the GAL promoter
|
K1: |
Dissociation constant for the GAL promoter |
T: |
Time constant representing rate of cellular division |
Equation 2
![]() |
(2) |
This is the equation for the rate of change of protein that is translated from the mRNA for GFP. The three terms represent production, degradation, and dilution respectively.
[M1] is the concentration of mRNA that translates the N-peptide GFP.
[GFP] represents the concentration of N-peptide and GFP.
[CFP] represents the concentration of the MS2-protein and CFP.
Parameter: |
Description |
λ2: |
Constant representing rate of translation of the mRNA that encodes for the production of N-peptide and GFP |
μ2: |
Constant representing rate of degradation of the GFP |
n2: |
Hill coefficient of the CFP/MS2 stem loop association
|
K2: |
Dissociation constant for the MS2-CFP protein to MS2 loop |
T: |
Time constant representing rate of cellular division |
Equation 3
![]() |
(3) |
This is the equation for the rate of change of the mRNA that is transcribed from the copper promoter. The three terms represent production, degradation, and dilution respectively.
[Cu2+] is the concentration of the copper added to the system that binds to the CUP1 promoter and activates the transcription of M2.
[M2] represents the concentration of mRNA that translates the MS2-protein and CFP.
Parameter |
Description |
λ3: |
Constant representing rate of transcription of the DNA that encodes for the production of the MS2-protein and CFP |
μ3: |
Constant representing rate of degradation of mRNA |
n3: |
Hill coefficient of the association between copper and the CUP1 promoter
|
K3: |
Dissociation constant for Copper promoter |
T: |
Time constant representing rate of cellular division |
Equation 4
![]() |
(4) |
This is the equation for the rate of change of protein that is translated from the mRNA for CFP. The three terms represent production, degradation, and dilution respectively.
[M2] is the concentration of mRNA that translates to MS2-protein and CFP.
[GFP] represents the concentration of the N-peptide and GFP.
[CFP] represents the concentration of the MS2-protein and CFP.
Parameters |
Description |
λ4: |
Constant representing rate of translation of the mRNA that encodes for the production of MS2-protein and CFP |
μ4: |
Constant representing rate of degradation of the CFP |
n4: |
Hill coefficient of the GFP/Bbox stem loop association
|
K4: |
Dissociation constant for the N-Pep-GFP protein to the Bbox-stem loop |
T: |
time constant representing rate of cellular division |
Parameter Study
The parameter values were first estimated based on the literature [1] and after the first estimation, a possible range of variation for each parameter was assigned, also based on literature. Then, we studied the bistability of the model depending on the parameter values that were varied in the above mentioned ranges. For more information, see Parameter Space Analysis and Directed Evolution.
Modification of the construct
Some experimental difficulties were encountered with the copper construct which led to the use of a methionine promoter to substitute it. Methionine acts as an inhibitor of the promoter, so that equation 3 had to be substituted by the following equation:
![](https://static.igem.org/mediawiki/2010/f/f2/Meth.png)
![](https://static.igem.org/mediawiki/2010/7/75/MET_toggle_switch.png)
The behaviour of the switch can then be summarise in the following table:
What is present in the system |
Protein(s) produced |
Galactose and Methionine |
GFP |
Galactose only |
GFP, CFP (doses dependent) |
Methionine only |
No GFP or CFP |
No Galactose and no Methionine |
CFP |
References
[1] Beyer A, Hollunder J, Nasheuer HP, Wilhelm T. (2004), Post-transcriptional expression regulation in the yeast Saccharomyces cerevisiae on a genomic scale, Mol Cell Proteomics., Vol. 3, No.11, pp. 1083-1092.
[2] Alon, U. (2006), An Introduction to Systems Biology: Design Principles of Biological Circuits, Chapman and Hall.
![]() |
Continue to Nullclines and Fixed Points ![]() |