Team:Aberdeen Scotland/parameters/invest 1

From 2009.igem.org

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== Dissociation Constants Continued ==
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Upon further investigation we have concluded that the vast majority of K<sub>d</sub> values found in papers were inaccurately low for the following reasons:
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1. Most K<sub>d</sub> values are measured in vitro, which gives a falsely low measurement since the conditions in the  reaction (most notable being salt concentration and pressure) are completely different than in an e. coli cell. The salt concentration affects the reaction significantly since it lowers the electrostatic affinity of the protein to the operon. We know from thermodynamics that pressure and temperature will change reaction kinetics and hence the in vitro experiments will different reaction rates and hence different K<sub>d</sub> values than the true behaviour in the cell.
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2. We have found measurements of K<sub>d</sub> values which have been done in conditions which try to replicate in vivo conditions. These K<sub>d</sub> values are better, but also far too low, since they do not take into account non-specific DNA binding and cell pressure.
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3. In our model, we are working in molecules per cell, instead of molarity. Upon converting the K<sub>d</sub> values from molarity to molecules per cell we found that a great deal the K<sub>d</sub> values we found were less than 1 molecule per cell. This implies that less than one protein (lacI, tetR etc) is needed to half the production. This is physically infeasible, the probability that one protein molecule will collide with a single operon in the cell at the correct angle is close to zero.
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We consulted with Prof. Peter McGlynn of the Institute of Medical Sciences in Aberdeen, who agreed with our analysis of the K<sub>d</sub> values and introduced the idea of non-specific DNA binding. He showed us a PhD thesis from one of his students Bryony Payne from 2006. In this thesis a far more direct and accurate measurement of the lacO repression was made. It stated that 340 tetremers of LacI was required to fully repress the lacO operon. From this value, we estimated the Kd value for the lacO operon to be 700 molecules per cell.
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=== Our new estimations for K<sub>d</sub>===
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Starting our estimation from Prof. Mcglynn’s PhD student Bryony Payne; 2006, we are given that 340 LacI tetramers completely repress a promoter. Hence roughly 120 tetramers will give half repression. Assuming the tetramers are stable this gives a value of K<sub>LacI</sub> , K<sub>d</sub> for LacI to LacO, of  4×170 or K<sub>LacI</sub> ~ 700.
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For the LacI IPTG complex formation, we estimated K<sub>IPTG</sub> ~1200 using [7] and our value of K<sub>LacI</sub> above.
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TetR to TetO seems to have a lower affinity to each other than LacI to LacO. However, the in vitro values suggest that TetR binds still with a strong affinity to TetR. Thus the K<sub>TetR</sub> value was roughly estimated to be up to 10 times K<sub>LacI</sub>.
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The in vitro values for cI to its operon seem to suggest that the in vivo K<sub>cI</sub> value is in the same order of magnitude, but possibily smaller, than K<sub>TetR</sub>.
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So we now have:
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K<sub>LacI</sub> = 700 molecules per cell
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K<sub>cI</sub> = 7000 molecules per cell
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K<sub>TetR</sub> = 7000 molecules per cell
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Revision as of 09:01, 10 August 2009

University of Aberdeen iGEM 2009

Contents

Dissociation Constants

Introduction

Our model uses hill kinetics; we have three repression hill functions of the form:

Dissociation Constants Eq 1.gif

It also has one activation hill function of the form:

Dissociation Constants Eq 2.gif

And one repression / induction hill function of the form

Dissociation Constants Eq 3.gif

Where β is the maximal transcription rate, [X] is the concentration of protein X and Kd is the dissociation constant for molecule X to the operator in question, [S] is the concentration of the inducer, S and Ks is the dissociation constant for the inducer to the repressor, X. Kd is defined as follows:

Dissociation Constants Eq 4.gif

Where koff and kon are the on and off rates in the equation

Dissociation Constants Eq 5.gif

Kd has a more biologically meaningful definition however, it is the concentration of X at which the operator will be repressed 50% of the time.

The issue

The units of Kd are usually given in M, the molarity, or moles per litre. Our model works with the exact number of molecules so we convert our Kd values into molecules per cell. This is achieved as follows:

Dissociation Constants Eq 6.gif

Where the volume of the cytoplasm of the cell is 6.7×10-16 litres

This conversion constant of Avogadro’s number multiplied by the cytoplasm volume is ~ 402000000 (402 million).

The problem with this is that most dissociation constants found in the literature equate to a value of molecules per cell that is less than 1. Clearly in a cell with 10 plasmids and therefore 10 operators 1 molecule could not repress all of them.

Below is a table of the conflicting information we found. This is an extract from the EHTZ wiki [6] with the new column of the value in molecules per cell added.


Parameter Value Value (molecules per cell) Description
KLacI 0.1 - 1 [pM] OR 800 [nM] 0.00004-0.0004 molecules OR 322 molecules LacI repressor dissociation constant
KIPTG 1.3 [µM] 522 molecules IPTG-LacI repressor dissociation constant
KtetR 179 [pM] 0.07 molecules TetR repressor dissociation constant
KCI 8 [pM] OR 50 [nM] 0.003 molecules OR 20 molecules CI repressor dissociation constant
KAHL 0.09 - 1 [µM] 402 molecules AHL-LuxR activator dissociation constant

And here are the other parameters we found in the literature

Parameter Value Value (molecules per cell) Description Reference
KLacI ~1*10 -12 M OR ~1.8*10-12 M 0.0004 molecules OR 0.00072 molecules Dissociation constant for LacI to LacO DNA site [1][2]
KIPTG 1*10-6 M 402 molecules Dissociation constant for IPTG to LacI [3]
KtetR (5.6 ± 2) × 10-9 M OR 1.53*10-8 M 2.25 molecules OR 6.1506 molecules Dissociation constant for TetR to TetO [4][5]
KCI 50 * 10-9 M 20 molecules Dissocitation constant for cI to DNA site [6]

Dissociation Constants Continued

Upon further investigation we have concluded that the vast majority of Kd values found in papers were inaccurately low for the following reasons:

1. Most Kd values are measured in vitro, which gives a falsely low measurement since the conditions in the reaction (most notable being salt concentration and pressure) are completely different than in an e. coli cell. The salt concentration affects the reaction significantly since it lowers the electrostatic affinity of the protein to the operon. We know from thermodynamics that pressure and temperature will change reaction kinetics and hence the in vitro experiments will different reaction rates and hence different Kd values than the true behaviour in the cell.

2. We have found measurements of Kd values which have been done in conditions which try to replicate in vivo conditions. These Kd values are better, but also far too low, since they do not take into account non-specific DNA binding and cell pressure.

3. In our model, we are working in molecules per cell, instead of molarity. Upon converting the Kd values from molarity to molecules per cell we found that a great deal the Kd values we found were less than 1 molecule per cell. This implies that less than one protein (lacI, tetR etc) is needed to half the production. This is physically infeasible, the probability that one protein molecule will collide with a single operon in the cell at the correct angle is close to zero.

We consulted with Prof. Peter McGlynn of the Institute of Medical Sciences in Aberdeen, who agreed with our analysis of the Kd values and introduced the idea of non-specific DNA binding. He showed us a PhD thesis from one of his students Bryony Payne from 2006. In this thesis a far more direct and accurate measurement of the lacO repression was made. It stated that 340 tetremers of LacI was required to fully repress the lacO operon. From this value, we estimated the Kd value for the lacO operon to be 700 molecules per cell.

Our new estimations for Kd

Starting our estimation from Prof. Mcglynn’s PhD student Bryony Payne; 2006, we are given that 340 LacI tetramers completely repress a promoter. Hence roughly 120 tetramers will give half repression. Assuming the tetramers are stable this gives a value of KLacI , Kd for LacI to LacO, of 4×170 or KLacI ~ 700.

For the LacI IPTG complex formation, we estimated KIPTG ~1200 using [7] and our value of KLacI above. TetR to TetO seems to have a lower affinity to each other than LacI to LacO. However, the in vitro values suggest that TetR binds still with a strong affinity to TetR. Thus the KTetR value was roughly estimated to be up to 10 times KLacI. The in vitro values for cI to its operon seem to suggest that the in vivo KcI value is in the same order of magnitude, but possibily smaller, than KTetR.

So we now have:

KLacI = 700 molecules per cell

KcI = 7000 molecules per cell

KTetR = 7000 molecules per cell

References

[1] Mitchel Lewis (2005) The Lac repressor. C. R. Biologies 328 (2005) 521–548

[2] Falcon C.M and Matthews K.S. (2000) Operator DNA sequence Variation Enhances High Affinity Binding by Hinge Helix Mutants of Lactose Repressor Protein. Biochemistry. 39, 11074-11084

[3] Uri Alon, An introduction to systems Biology, p244

[4] Nucleic Acids Res. 2004; 32(2): 842–847. Two mutations in the tetracycline repressor change the inducer anhydrotetracycline to a corepressor Annette Kamionka, Joanna Bogdanska-Urbaniak, Oliver Scholz, and Wolfgang Hillen*

[5] Volume 272, Number 11, Issue of March 14, 1997 pp. 6936-6942, The Role of the Variable Region in Tet Repressor for Inducibility by Tetracycline, Christian Berens , Dirk Schnappinger and Wolfgang Hillen

[6] http://parts.mit.edu/igem07/index.php?title=ETHZ/Parameters

[7] Detailed map of a cis-regulatory input function – Y. Setty*,†, A. E. Mayo*,†, M. G. Surette‡, and U. Alon*,†,§