IGEM:Imperial/2010/Michaelis Menten: Difference between revisions
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=Model based on Michaelis Menten Kinetics (Weeks 4 and 5)= | =Model based on Michaelis Menten Kinetics (Weeks 4 and 5)= | ||
Motivation | ==Motivation== | ||
We came up with a simple concept of output amplification, which is enhanced by using enzymes. It is beneficial for us to model the behaviour of our design so that we will be able to answer the following questions. | |||
# How beneficial is the use of amplification? (Compare speed of response of transcription (and translation) with 1- or 2-step amplification) | |||
# How beneficial is the use of amplification? ( | # How many amplification steps are beneficial to have? Will further adding of amplification steps introduce too many time delays? | ||
# How many amplification steps are beneficial to have? | # Is it better to use TEV all or HIV1? | ||
# | |||
Modelling should allows us to make a decision on which design is the most efficient one. | |||
==First Model== | ==First Model== | ||
===HIV1=== | ===HIV1=== | ||
{| class="wikitable" style="text-align: center; width: 100%; height: 170px;" | {| class="wikitable" style="text-align: center; width: 100%; height: 170px;" | ||
| [[Image:Slide2.JPG|300px|thumb|center|alt=A|At each stage of amplification a distinct protease is being used ]] | |||
| align="left"| | |||
*m | '''Equations''' | ||
*p_h | |||
*<math>\dot{m}=k_{to} - d_{to}m</math> | |||
*<math>\dot{p_h} = k_hm - d_hp_h</math> | |||
*<math>\dot{p_t} = k_tp_h - d_tp_t</math> | |||
*<math>\dot{p_g} = k_gp_t - d_gp_g</math> | |||
| align="left"| | |||
'''Parameters''' | |||
*<math>k_{to}\mbox{...transcription rate of HIV1}</math> | |||
*<math>d_{to}\mbox{...degradation rate of mRNA coding for HIV1}</math> | |||
*<math>k_h\mbox{...translation rate of HIV1}</math> | |||
*<math>d_h\mbox{...degradation rate of HIV1}</math> | |||
*<math>k_t\mbox{...production rate of TEV by HIV1}</math> | |||
*<math>d_t\mbox{...degradation rate of TEV}</math> | |||
*<math>k_g\mbox{...production rate of GFP by TEV}</math> | |||
*<math>d_g\mbox{...degradation rate of GFP}</math> | |||
|} | |} | ||
===TEV=== | ===TEV=== | ||
{| class="wikitable" style="text-align: center; width: 100%; height: 170px;" | {| class="wikitable" style="text-align: center; width: 100%; height: 170px;" | ||
|[[Image:TEV.jpg|300px|thumb|center|alt=A|TEV is used at both stages of amplification]] | |||
|align="left"| | |||
'''Equations''' | '''Equations''' | ||
*m | |||
*p_t | |||
* | *<math>\dot{m} = k_{to} - d_{to}m</math> | ||
*p_g | |||
'''Parameters''' | *<math>\dot{p_t} = k_tm - d_tp_t</math> | ||
* | |||
* | |||
*k_t...rate of translation of TEV | *<math>\dot{p_{ts}} = k_{ts}p_t - d_{ts}p_{ts}</math> | ||
*d_t...degradation rate of TEV | |||
* | |||
* | *<math>\dot{p_g} = k_{g1}p_t + k_{g2}p_{ts} - d_gp_g</math> | ||
* | |align="left"| | ||
* | '''Parameters''' | ||
*d_g...degradation rate of GFP | *<math>k_{to}\mbox{...rate of transcription by TEV}</math> | ||
*<math>d_{to}\mbox{...degradation rate of mRNA coding for TEV}</math> | |||
*<math>k_t\mbox{...rate of translation of TEV}</math> | |||
*<math>d_t\mbox{...degradation rate of TEV}</math> | |||
*<math>k_{ts}\mbox{...rate of production (fusion) of split TEV}</math> | |||
*<math>d_{ts}\mbox{...degradation rate of split TEV}</math> | |||
*<math>k_{g1}\mbox{...rate of production of GFP by full TEV}</math> | |||
*<math>k_{g2}\mbox{...rate of production of GFP by split TEV}</math> | |||
*<math>d_g\mbox{...degradation rate of GFP}</math> | |||
|} | |} | ||
==Improved Model== | ==Improved Model which accounts for enzyme reactions (28/07/2010)== | ||
[[Image: | ===TEV=== | ||
{| class="wikitable" style="text-align: center; width: 100%; height: 170px;" | |||
|[[Image:TEV.jpg|500px|thumb|center|alt=A|TEV is used at both stages of amplification]] | |||
|align="left"| | |||
'''Equations''' | |||
* 1. Production of TEV from transcription | |||
<math>\dot{p_t} = s_t - d_tp_t</math> | |||
<math>s_t = \dfrac{k_tk_{to}}{d_{to}}</math> | |||
* 2. Production of split TEV from transcription | |||
<math>\dot{p_{st}} = s_{st} - d_{st}p_{st}</math> | |||
* 3. Production of split GFP from transcription | |||
<math>\dot{p_{sg}} = s_{sg} - d_{sg}p_{sg}</math> | |||
* 4. Production of fused split TEV catalysed by TEV (1) | |||
<math>\dot{p_{ts}} = \dfrac{V_{max,t}[p_{st}]}{K_{m,ts} + [p_{st}]} - d_{ts}p_{ts}</math> | |||
* 5. Production of GFP catalysed by TEV (1) and fused split TEV (4) | |||
<math>\dot{p_g} = \dfrac{V_{max,tg}[p_{sg}]}{K_{m,tg} + [p_{sg}]} + \dfrac{V_{max,tsg}[p_{sg}]}{K_{m,tsg} + [p_{sg}]} - d_gp_g</math> | |||
|} | |||
===Implementation in Matlab=== | ===Implementation in Matlab=== | ||
Line 61: | Line 103: | ||
{| border="1" | {| border="1" | ||
! | ! | ||
! GFP | ! GFP | ||
! TEV | ! TEV | ||
Line 67: | Line 109: | ||
! split GFP | ! split GFP | ||
|- | |- | ||
|Km and | |'''<math>Km</math> and <math>k_{cat}</math>''' | ||
| | | - | ||
|[http://peds.oxfordjournals.org/cgi/reprint/14/12/993 | |<math>K_m = 0.061</math>; <math>k_{cat} = 0.16</math>; [http://peds.oxfordjournals.org/cgi/reprint/14/12/993] | ||
| 40% of | | 40% of value for TEV | ||
| | | - | ||
|- | |- | ||
| | | '''Half-life or degradation rate''' | ||
| Half-life | | Half-life in B.sub approximately 1.5 hours | ||
| ? | | ? | ||
| ? | | ? | ||
| Half-life shorter than GFP | | Half-life shorter than GFP | ||
|- | |- | ||
| | | '''Production rate in B.sub''' | ||
| ? | | ? | ||
| ? | | ? | ||
Line 88: | Line 130: | ||
==Conclusion== | ==Conclusion== | ||
We | We were not able to obtain all the necessary constants. Hence, we decided to make educated guesses about possible relative values between the constants as well as varying them and observing the change in output. | ||
As the result, we concluded that the amplification happens at each amplification level proposed. The magnitude of amplification varies depending on the constants. There is not much difference between using TEV or HIV1. | |||
==References== | |||
#Kapust R. et al (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Engineering. [Online] 14(12), 993-1000. Available from: http://peds.oxfordjournals.org/cgi/reprint/14/12/993 [Accessed 28th July 2010] |
Latest revision as of 16:16, 21 October 2010
Model based on Michaelis Menten Kinetics (Weeks 4 and 5)
Motivation
We came up with a simple concept of output amplification, which is enhanced by using enzymes. It is beneficial for us to model the behaviour of our design so that we will be able to answer the following questions.
- How beneficial is the use of amplification? (Compare speed of response of transcription (and translation) with 1- or 2-step amplification)
- How many amplification steps are beneficial to have? Will further adding of amplification steps introduce too many time delays?
- Is it better to use TEV all or HIV1?
Modelling should allows us to make a decision on which design is the most efficient one.
First Model
HIV1
Equations
|
Parameters
|
TEV
Equations
|
Parameters
|
Improved Model which accounts for enzyme reactions (28/07/2010)
TEV
Equations
[math]\displaystyle{ \dot{p_t} = s_t - d_tp_t }[/math] [math]\displaystyle{ s_t = \dfrac{k_tk_{to}}{d_{to}} }[/math]
[math]\displaystyle{ \dot{p_{st}} = s_{st} - d_{st}p_{st} }[/math]
[math]\displaystyle{ \dot{p_{sg}} = s_{sg} - d_{sg}p_{sg} }[/math]
[math]\displaystyle{ \dot{p_{ts}} = \dfrac{V_{max,t}[p_{st}]}{K_{m,ts} + [p_{st}]} - d_{ts}p_{ts} }[/math]
[math]\displaystyle{ \dot{p_g} = \dfrac{V_{max,tg}[p_{sg}]}{K_{m,tg} + [p_{sg}]} + \dfrac{V_{max,tsg}[p_{sg}]}{K_{m,tsg} + [p_{sg}]} - d_gp_g }[/math] |
Implementation in Matlab
The Matlab code for the different stages of amplification and diagrams can be found here.
Kinetic constants
GFP | TEV | split TEV | split GFP | |
---|---|---|---|---|
[math]\displaystyle{ Km }[/math] and [math]\displaystyle{ k_{cat} }[/math] | - | [math]\displaystyle{ K_m = 0.061 }[/math]; [math]\displaystyle{ k_{cat} = 0.16 }[/math]; [1] | 40% of value for TEV | - |
Half-life or degradation rate | Half-life in B.sub approximately 1.5 hours | ? | ? | Half-life shorter than GFP |
Production rate in B.sub | ? | ? | ? | ? |
Conclusion
We were not able to obtain all the necessary constants. Hence, we decided to make educated guesses about possible relative values between the constants as well as varying them and observing the change in output.
As the result, we concluded that the amplification happens at each amplification level proposed. The magnitude of amplification varies depending on the constants. There is not much difference between using TEV or HIV1.
References
- Kapust R. et al (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Engineering. [Online] 14(12), 993-1000. Available from: http://peds.oxfordjournals.org/cgi/reprint/14/12/993 [Accessed 28th July 2010]