Team:LCG-UNAM-Mexico/Notebook/2008-September

From 2008.igem.org

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<td class="bodyText"><p><br />
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  <strong>Converting units </strong><br />
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  <br />
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  <strong>1. Reaction 1. </strong><br />
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  3.723mM =? Molecules <br />
 +
  <br />
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  How to convert from Molar to Mole? <br />
 +
  M = mole / liter <br />
 +
  -&gt; What is the volume (in liters) of a cell? <br />
 +
&lt;&lt;The volume of a bacterium is 10^-15L&gt;&gt; Molar concentration. Example. Wikipedia. <br />
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<br />
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3.723mM = 37.23x10-18 mol at 10-15  liters <br />
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37.23 x10-18 mol = 224.20427x105 molecules <br />
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<br />
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*  1 mol = 6.02214x1023 molecules <br />
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<strong><br />
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2. Reaction 6. </strong><br />
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<br />
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The flow in 20 plasmids is 20mM / h ... <br />
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-&gt; in 10 plasmids, 10mM / h <br />
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<br />
 +
Flow = 10 mM / h = 10mM/3600s = 0.00278mM / s <br />
 +
       
 +
but ... 0.00278mM = 0.0278x10-18mol in 10-15 liters <br />
 +
       
 +
... 0.0278x10-18mol = 1.67415x105 molecules. <br />
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<br />
 +
The flow in the cell is 1.67415x104 molecules / s with 10 copies (plasmids). <br />
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ν = k * [promoter] <br />
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1.67415x104 molecules / s = k * 10 molecules <br />
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-&gt; k = 1.67415x103 molecules / s <br />
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<br />
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<strong>3. Reaction 5. </strong></p>
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<p>  ΔG ° =- 23.81 kcal / mol <br />
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  Keq = exp (-ΔG º / RT) <br />
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  <br />
 +
  -&gt; The units do not affect using this formula! <br />
 +
  Is it correct? <br />
 +
  <strong><br />
 +
  Correction of the synthesis reaction of cI </strong><br />
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  <br />
 +
  Units of the k3ON, estimated in reference 3 are molecules-1seconds-1, which means that the reaction is second order. <br />
 +
  <br />
 +
  In that same article, they suggest that the mere presence of the dimer ensures  the production of cI with k3ON rate (that is, that bonding is  efficient). <br />
 +
  <br />
 +
  Since the estimated values do not consider the  intermediate step of the promoter's union and the complex, we should not  consider it. <br />
 +
  <br />
 +
  3. ... <br />
 +
  3.1 ρcI + (AHL: LuxR): (AHL: LuxR) -&gt; CI + ρcI + (AHL: LuxR): (AHL: LuxR) <br />
 +
  k3ON <br />
 +
  <strong><br />
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  Unknown parameters </strong><br />
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  <br />
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  </p>
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<table width="266" border="2">
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  <tr>
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    <td width="147">cI Dimerization</td>
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    <td width="101"> k4.1 &amp; k-4.1</td>
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  </tr>
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  <tr>
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    <td>Suppression by CI</td>
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    <td>V5max or k5 </td>
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  </tr>
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  <tr>
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    <td>Nickel Extrusion </td>
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    <td>k7</td>
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  </tr>
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  <tr>
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    <td>RcnA Degradation </td>
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    <td> k8</td>
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  </tr>
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  <tr>
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    <td>Nickel Internalization </td>
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    <td><p>k9</p>
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    </td>
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  </tr>
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</table>
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<p><br /></p>
         </td>
         </td>
       </tr>   
       </tr>   

Revision as of 03:09, 15 October 2008

LCG-UNAM-Mexico:Notebook/September

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September

2008-09-3


Lac promoter synthesis rate

van Hoek F, Hogeweg P (2007) The effect of stochasticity on the Lac Operon: An evolutionary perspective. PLoS Comput Biol 3 (6): E111

The article's objective, as you can infer from the title, is to evaluate the effect of stochasticity in the evolution of a Promoter. In order to do so they built a comprehensive model including every parameter involved in Transcription and translation. They measure some parameters but they depend mostly on literature to define them.

They both do a deterministic and a Stochastic analysis. To generate a Stochastic model they added one parameter, the average burst size of protein translation (protein translation occurs in bursts, after a several mRNA is synthesized proteins can be translated from the same mRNA). This was possible because when an mRNA molecule is translated it can not be degraded. Therefore after each translation it can either be translated again (p) or be degraded (1-p). This suggests that protein production occurs in bursts with a burst size geometrically distributed. After they compared their noise levels in the model with the experimental noise measurements they found good agreement.

Transcription they used to model a two-dimensional Hill-function dependent on the cAMP concentration and alloctase. (repressa the glucose and lactose Operon via cAMP activates the Operon via allolactose).

They use 11 biochemical parameters, including three of special importance for us:

a, when the rate Transcription RNA Polymerase is bound to the DNA, but CRP and Laci are not. evolva: initial value: 1.1 × 10-7 mM / min

b, The Transcription rate when both RNA Polymerase and CRP are bound, but Laci is not bound to the DNA. evolva: initial value: 2.2 × 10-5 mM / min

c,''leakiness,''the Transcription rate when RNA Polymerase is not bound to the DNA. evolva: initial value 5.5 × 10-10 mM / min

They modele binomially protein degradation, assuming that cell when to divide proteins are divided randomly between the cells. However in a population of non-Dividing cells this "dilution" can not be taken in account.

2008-09-17

-

2008-09-18

-

2008-09-19


Converting units

1. Reaction 1.
3.723mM =? Molecules

How to convert from Molar to Mole?
M = mole / liter
-> What is the volume (in liters) of a cell?
<<The volume of a bacterium is 10^-15L>> Molar concentration. Example. Wikipedia.

3.723mM = 37.23x10-18 mol at 10-15 liters
37.23 x10-18 mol = 224.20427x105 molecules

* 1 mol = 6.02214x1023 molecules

2. Reaction 6.


The flow in 20 plasmids is 20mM / h ...
-> in 10 plasmids, 10mM / h

Flow = 10 mM / h = 10mM/3600s = 0.00278mM / s
        but ... 0.00278mM = 0.0278x10-18mol in 10-15 liters
        ... 0.0278x10-18mol = 1.67415x105 molecules.

The flow in the cell is 1.67415x104 molecules / s with 10 copies (plasmids).
ν = k * [promoter]
1.67415x104 molecules / s = k * 10 molecules
-> k = 1.67415x103 molecules / s

3. Reaction 5.

ΔG ° =- 23.81 kcal / mol
Keq = exp (-ΔG º / RT)

-> The units do not affect using this formula!
Is it correct?

Correction of the synthesis reaction of cI


Units of the k3ON, estimated in reference 3 are molecules-1seconds-1, which means that the reaction is second order.

In that same article, they suggest that the mere presence of the dimer ensures the production of cI with k3ON rate (that is, that bonding is efficient).

Since the estimated values do not consider the intermediate step of the promoter's union and the complex, we should not consider it.

3. ...
3.1 ρcI + (AHL: LuxR): (AHL: LuxR) -> CI + ρcI + (AHL: LuxR): (AHL: LuxR)
k3ON

Unknown parameters


cI Dimerization k4.1 & k-4.1
Suppression by CI V5max or k5
Nickel Extrusion k7
RcnA Degradation k8
Nickel Internalization

k9


2008-09-22


Dimerization of cI

k4.1 & k-4.1?

2 cI <-> cI: cI

¿Quasi-balance?
The initial concentration of cI varies over time, but the proportion is preserved.
... Avoid kinetic analysis of the fast reactions, ie to take into consideration only their equilibrium constants instead of considering their rates (Kholodenko et al. 1998).

Keq = [cI: cI] / [cI] [cI] -> [cI:cI] = Keq [cI] 2

d (cI:cI 2cI) / dt = v+ - v -
d (Keq [cI]^2 + 2cI) / dt = v+ - v-

2008-09-23



General checkup

Simulation: The previous crisis was overcome... We are walking fine, however, there are still some parameters missing, but it has been decided that they will be estimated by adjusting the model and experimentally when we have the opportunity.

Data: There are a few missing parameters and we know that most of them are not available, so we have given up the search... What has yet to be defined in terms of data are concentrations of AiiA and LuxR, considering that they are preserved constants in the model.

We will also design experiments to obtain the missing parameters through the experimental measurements (viable).

Analysis: Stechiometric matrix: There was a meeting today in the morning with Osbaldo to review its analysis, we will share the information as soon as possible.

Sensitivity analysis: Although we don't understand yet the particular units in which SimBiology returns the results, the first graphics that show the basic parameters of the model are ready and they are the ones involved in the degradation of AHL and dimerization of it with LuxR ( the start of the cascade) and the ones regarding the entry and exit of Nickel. We must do this analysis recurrently, as we have seen that this is sensitive to the initial concentrations of metabolites, which are not yet fully defined.

States and stationary Jacobian: They were stopped briefly because we need the parameters for further analysis.  

Experimental

Requirements: Urgent! We need to send the oligos required to synthesis; We are working on it, but let's consider this a priority.

Electrodes: The device is not ready.

Design of experiments: The first meeting will be today.

Funds & Jambouree: We are still waiting for some sponsors to reply.