Team:KULeuven/Model/Reset

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<div style="float: right;">[[Image:logo_reset.jpg|120px]]</div>
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== Pulse Generator ==
== Pulse Generator ==
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| 7.0E-4 s<sup>-1</sup>
| 7.0E-4 s<sup>-1</sup>
|  
|  
-
| [http://parts.mit.edu/igem07/index.php?title=ETHZ/Parameters link]
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|  
|-
|-
| d<sub>RNA_Lac</sub>
| d<sub>RNA_Lac</sub>
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| 0.00337
| 0.00337
| binding cI on cI-Promotor
| binding cI on cI-Promotor
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| [http://parts.mit.edu/igem07/index.php?title=ETHZ/Parameters link]
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|  
|-
|-
! colspan="4" style="border-bottom: 1px solid #003E81;" | Transcription rates
! colspan="4" style="border-bottom: 1px solid #003E81;" | Transcription rates
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| 0.167 s<sup>-1</sup>
| 0.167 s<sup>-1</sup>
| RBS is B0032 (efficiency 0.3)
| RBS is B0032 (efficiency 0.3)
-
| [http://partsregistry.org/Part:BBa_B0032 link]
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|  
|-
|-
! colspan="4" style="border-bottom: 1px solid #003E81;" | Hill cooperativity
! colspan="4" style="border-bottom: 1px solid #003E81;" | Hill cooperativity
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| 2.0
| 2.0
|  
|  
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| [http://parts.mit.edu/igem07/index.php?title=ETHZ/Parameters link]
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|  
|}
|}
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<br>
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All references for parameters can be found below, in the new section.
=== Models ===
=== Models ===
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<body>
<body>
<p>
<p>
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<a href="https://static.igem.org/mediawiki/2008/6/6a/Lactonaseproduction.pdf">
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<a href="https://static.igem.org/mediawiki/2008/e/e7/LactonaseProductionODE.pdf">
<img border="0" src="https://2008.igem.org/wiki/skins/common/images/icons/fileicon-pdf.png" width="65" height="60">
<img border="0" src="https://2008.igem.org/wiki/skins/common/images/icons/fileicon-pdf.png" width="65" height="60">
</a>
</a>
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! colspan="4" style="border-bottom: 1px solid #003E81;" | Degradation rates
! colspan="4" style="border-bottom: 1px solid #003E81;" | Degradation rates
|-
|-
-
| d<sub>lactonase</sub>
+
| d<sub>aiiA</sub>
| | d<sub>LVA</sub> = 2.814E-4 s<sup>-1</sup>
| | d<sub>LVA</sub> = 2.814E-4 s<sup>-1</sup>
| LVA-tag reduces lifetime to 40 minutes
| LVA-tag reduces lifetime to 40 minutes
-
| [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=106306 link]
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| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [3<html>]</html>]
|-
|-
-
| d<sub>closed mRNA lactonase</sub>
+
| d<sub>closed mRNA aiiA</sub>
| 0.0046209812 s<sup>-1</sup>
| 0.0046209812 s<sup>-1</sup>
| estimate: because this RNA isn't translated, it degrades faster
| estimate: because this RNA isn't translated, it degrades faster
-
| [http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=124983&blobtype=pdf link]
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| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [2<html>]</html>]
|-
|-
-
| d<sub>open mRNA lactonase</sub>
+
| d<sub>open mRNA aiiA</sub>
| 0.0023104906 s<sup>-1</sup>
| 0.0023104906 s<sup>-1</sup>
|  
|  
-
| [http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=124983&blobtype=pdf link]
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| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [2<html>]</html>]
|-
|-
-
| d<sub>open mRNA lactonase complex</sub>
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| d<sub>mRNA aiiA complex</sub>
| 0.0023104906 s<sup>-1</sup>
| 0.0023104906 s<sup>-1</sup>
|  
|  
-
| [http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=124983&blobtype=pdf link]
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| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [2<html>]</html>]
|-
|-
! colspan="4" style="border-bottom: 1px solid #003E81;" | T7 Transcription
! colspan="4" style="border-bottom: 1px solid #003E81;" | T7 Transcription
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| 421
| 421
| dissociation constant, recalculated to remove units
| dissociation constant, recalculated to remove units
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| [http://www.jbc.org/cgi/content/full/279/5/3239 link]
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| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [4<html>]</html>]
|-
|-
| k<sub>max</sub>
| k<sub>max</sub>
| 0.044 s<sup>-1</sup>
| 0.044 s<sup>-1</sup>
| maximal T7 transcription rate
| maximal T7 transcription rate
-
| [http://www.jbc.org/cgi/content/full/279/5/3239 link]
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| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [4<html>]</html>]
|-
|-
! colspan="4" style="border-bottom: 1px solid #003E81;" | Key-Lock constants
! colspan="4" style="border-bottom: 1px solid #003E81;" | Key-Lock constants
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| K<sub>eq 1</sub>
| K<sub>eq 1</sub>
| 0,015 [M]
| 0,015 [M]
-
| between closed and open Lactonase mRNA, experimental
+
| between closed and open Lactonase mRNA, modeled for competition, experimental
-
| [http://parts2.mit.edu/wiki/index.php/Berkeley2006-RiboregulatorsMain link]
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| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [1<html>]</html>]
|-
|-
| K<sub>eq 2</sub>
| K<sub>eq 2</sub>
| 0.0212 [M]
| 0.0212 [M]
-
| between closed Lactonase mRNA and key unlocked mRNA complex, experimental
+
| between closed Lactonase mRNA and key unlocked mRNA complex, modeled for competition, experimental
-
| [http://parts2.mit.edu/wiki/index.php/Berkeley2006-RiboregulatorsMain link]
+
| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [1<html>]</html>]
|-
|-
| k<sub>dis1</sub>
| k<sub>dis1</sub>
-
| 100 s<sup>-1</sup>
+
| 0.00416 s<sup>-1</sup>
-
| derived from experimental values
+
| estimate: derived from experimental values
-
| [http://parts2.mit.edu/wiki/index.php/Berkeley2006-RiboregulatorsMain link]
+
| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [1<html>]</html>]
|-
|-
| k<sub>complex1</sub>
| k<sub>complex1</sub>
-
| 57 s<sup>-1</sup>
+
| 0.00237 s<sup>-1</sup>
-
| derived from experimental values
+
| estimate: derived from experimental values
-
| [http://parts2.mit.edu/wiki/index.php/Berkeley2006-RiboregulatorsMain link]
+
| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [1<html>]</html>]
|-
|-
| k<sub>closed</sub>
| k<sub>closed</sub>
-
| 100 s<sup>-1</sup>
+
| 500 s<sup>-1</sup>
-
| derived from experimental values
+
| estimate: derived from experimental values
-
| [http://parts2.mit.edu/wiki/index.php/Berkeley2006-RiboregulatorsMain link]
+
| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [1<html>]</html>]
|-
|-
| k<sub>open</sub>
| k<sub>open</sub>
-
| 1.5 s<sup>-1</sup>
+
| 7.5 s<sup>-1</sup>
-
| derived from experimental values
+
| estimate: derived from experimental values
-
| [http://parts2.mit.edu/wiki/index.php/Berkeley2006-RiboregulatorsMain link]
+
| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [1<html>]</html>]
|-
|-
| k<sub>translation</sub>
| k<sub>translation</sub>
| 0.167 s<sup>-1</sup>
| 0.167 s<sup>-1</sup>
| lock defined translation rate for Lactonase
| lock defined translation rate for Lactonase
-
|  
+
| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [1<html>]</html>]
|}
|}
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=== Simulation ===
=== Simulation ===
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Remark: up to date with latest version?
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During the first 50000 seconds, the input signal TetR (no aTc) is held constant at 5E-5 s<sup>-1</sup>. This results in a background signal of lactonase which will convert a small part of the HSL into hydroxy acid. From 50000 till 80000 seconds, the input signal is at its maximum value: 0.0125 s<sup>-1</sup> (aTc added). This results in an increase of lactonase which converts almost every HSL molecule into the hydroxy acid. The timer is set to 0. After a delay of approximately 20000 seconds, the amount of HSL starts to increase again: the clock is ticking. A shorter pulse of TetR (1000 seconds) only partially resets the timer: not all the HSL is converted into hydroxy acid. The graph has amounts (number of molecules in the cell) plotted vs time, measured in seconds.
-
The number lactonase genes is held constant during the entire simulation. For the first 15.000 seconds the number of mRNA Ribokey is equal to 0.015 and the number of pT7 molecules to 0.4, then for 15000 s these numbers are set on 6 and 3 respectively (based on the results of the model of the filter) after which they are reduced back to 0.015 and 0.4. <br>
 
-
We see that an increase in the number of mRNA Ribokey and pT7(due to an increase in light intensity) will lead to a much higher number of lactonase molecules.
 
[[Image:Sim_lactonaseproduction_1.png|700px|center]]
[[Image:Sim_lactonaseproduction_1.png|700px|center]]
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=== References ===
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<head>
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<meta http-equiv="Content-Type" content="text/html; charset=utf-8"/>
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<title>Bibliography</title>
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<table style="border-collapse:collapse;line-height:1.1em;">
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<tr style="vertical-align:top;"><td>[1]</td><td style="padding-left:4pt;">“Berkeley2006-RiboregulatorsMain - IGEM”; http://parts2.mit.edu/wiki/index.php/Berkeley2006-RiboregulatorsMain.</td></tr>
 +
<tr><td colspan="2">&nbsp;</td></tr>
 +
<tr style="vertical-align:top;"><td>[2]</td><td style="padding-left:4pt;">J.A. Bernstein et al., “Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays,” <span style="font-style:italic;">Proceedings of the National Academy of Sciences of the United States of America</span>,  vol. 99, Jul. 2002, pp. 9697–9702. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1073/pnas.112318199&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Global%20analysis%20of%20mRNA%20decay%20and%20abundance%20in%20Escherichia%20coli%20at%20single-gene%20resolution%20using%20two-color%20fluorescent%20DNA%20microarrays&amp;rft.jtitle=Proceedings%20of%20the%20National%20Academy%20of%20Sciences%20of%20the%20United%20States%20of%20America&amp;rft.stitle=Proc%20Natl%20Acad%20Sci%20U%20S%20A.%20&amp;rft.volume=99&amp;rft.issue=15&amp;rft.aufirst=Jonathan%20A.&amp;rft.aulast=Bernstein&amp;rft.au=Jonathan%20A.%20Bernstein&amp;rft.au=Arkady%20B.%20Khodursky&amp;rft.au=Pei-Hsun%20Lin&amp;rft.au=Sue%20Lin-Chao&amp;rft.au=Stanley%20N.%20Cohen&amp;rft.date=2002-07-23&amp;rft.pages=9697%E2%80%939702"></span></td></tr>
 +
 +
<tr><td colspan="2">&nbsp;</td></tr>
 +
<tr style="vertical-align:top;"><td>[3]</td><td style="padding-left:4pt;">J.B. Andersen et al., “New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria,” <span style="font-style:italic;">Applied and Environmental Microbiology</span>,  vol. 64, Jun. 1998, pp. 2240–2246. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=New%20Unstable%20Variants%20of%20Green%20Fluorescent%20Protein%20for%20Studies%20of%20Transient%20Gene%20Expression%20in%20Bacteria&amp;rft.jtitle=Applied%20and%20Environmental%20Microbiology&amp;rft.stitle=Appl%20Environ%20Microbiol.%20&amp;rft.volume=64&amp;rft.issue=6&amp;rft.aufirst=Jens%20Bo&amp;rft.aulast=Andersen&amp;rft.au=Jens%20Bo%20Andersen&amp;rft.au=Claus%20Sternberg&amp;rft.au=Lars%20Kongsbak%20Poulsen&amp;rft.au=Sara%20Petersen%20Bj%C3%B8rn&amp;rft.au=Michael%20Givskov&amp;rft.au=S%C3%B8ren%20Molin&amp;rft.date=1998-06&amp;rft.pages=2240%E2%80%932246"></span></td></tr>
 +
<tr><td colspan="2">&nbsp;</td></tr>
 +
<tr style="vertical-align:top;"><td>[4]</td><td style="padding-left:4pt;">G.M. Skinner et al., “Promoter Binding, Initiation, and Elongation By Bacteriophage T7 RNA Polymerase: A SINGLE-MOLECULE VIEW OF THE TRANSCRIPTION CYCLE,” <span style="font-style:italic;">J. Biol. Chem.</span>,  vol. 279, Jan. 2004, pp. 3239-3244. <span class="Z3988" title="url_ver=Z39.88-2004&amp;ctx_ver=Z39.88-2004&amp;rft_id=info%3Adoi/10.1074/jbc.M310471200&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Promoter%20Binding%2C%20Initiation%2C%20and%20Elongation%20By%20Bacteriophage%20T7%20RNA%20Polymerase%3A%20A%20SINGLE-MOLECULE%20VIEW%20OF%20THE%20TRANSCRIPTION%20CYCLE&amp;rft.jtitle=J.%20Biol.%20Chem.&amp;rft.volume=279&amp;rft.issue=5&amp;rft.aufirst=Gary%20M.&amp;rft.aulast=Skinner&amp;rft.au=Gary%20M.%20Skinner&amp;rft.au=Christoph%20G.%20Baumann&amp;rft.au=Diana%20M.%20Quinn&amp;rft.au=Justin%20E.%20Molloy&amp;rft.au=James%20G.%20Hoggett&amp;rft.date=2004&amp;rft.pages=3239-3244"></span></td></tr>
 +
<tr><td colspan="2">&nbsp;</td></tr>
 +
</table></body>
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</html>

Latest revision as of 14:56, 3 October 2008

  dock/undock dropdown  

Logo reset.jpg

Contents

Pulse Generator

Position in the system

The Pulse Generator-subsystem is directly linked to the Filter.

When the filter indicates that the input is zero (there is no desease), the system will (ideally) produce no lactonase. As soon as the output of the filter is one, the subsystem will produce a pulse of lactonase which will be high enough to 'remove' all HSL present in the system and in that way reset the timer.

Describing the system

Pulse Generator BioBrick.jpg

ODE's

NOT AVAILABLE

Parameters

Parameter values (Pulse Generator)
Name Value Comments Reference
Degradation rates
dRNA_cI 0.00462 s-1
dcI 7.0E-4 s-1
dRNA_Lac 0.00231 s-1
dLac 2.888E-4 s-1
dRNA_Ribokey:cI 0.00231 s-1
Dissociation constants
KRibokey:cI 0.00212 kass/kdiss for the Ribokey cI complex
KcI 0.00337 binding cI on cI-Promotor
Transcription rates
kRNA_cI 0.025 s-1 maximal transcription rate RNA cI (no cI repressor present)
kRNA_Lac 0.025 s-1
Translation rates
kcI 0.167 s-1
kLac 0.167 s-1 RBS is B0032 (efficiency 0.3)
Hill cooperativity
ncI 2.0


All references for parameters can be found below, in the new section.

Models

CellDesigner (SBML file)

Pulse Generator

Matlab

Pulse Generator Matlab.jpg

Problem

The idea of a pulsgenerator as reset mechanism doesn't meet the black-box requirements for the following reasons:

  • it takes too long before the proposed system generates a pulse-like event
  • the pulse itself is too long
  • a constant lactonase production sequence generates enough lactonase to reset the timer

Constant Lactonase Production

Pictogram lactonaseproduction.png

Position in the system

The Constant Lactonase Production-system is directly linked to the Filter.

When the filter indicates that the input is zero (there is no desease), the system will (ideally) produce no lactonase. As soon as the output of the filter is one, the system starts producing lactonase and remains doing this untill the light goes off again. In this way all the HSL-molecules that are present will be 'removed' and the timer is reset.

Describing the system

see also: Project:Reset

Modeling Reset.PNG

ODE's

Parameters

Parameter values Constant Lactonase Production
Name Value Comments Reference
Degradation rates
daiiA dLVA = 2.814E-4 s-1 LVA-tag reduces lifetime to 40 minutes [3]
dclosed mRNA aiiA 0.0046209812 s-1 estimate: because this RNA isn't translated, it degrades faster [2]
dopen mRNA aiiA 0.0023104906 s-1 [2]
dmRNA aiiA complex 0.0023104906 s-1 [2]
T7 Transcription
KT7 421 dissociation constant, recalculated to remove units [4]
kmax 0.044 s-1 maximal T7 transcription rate [4]
Key-Lock constants
Keq 1 0,015 [M] between closed and open Lactonase mRNA, modeled for competition, experimental [1]
Keq 2 0.0212 [M] between closed Lactonase mRNA and key unlocked mRNA complex, modeled for competition, experimental [1]
kdis1 0.00416 s-1 estimate: derived from experimental values [1]
kcomplex1 0.00237 s-1 estimate: derived from experimental values [1]
kclosed 500 s-1 estimate: derived from experimental values [1]
kopen 7.5 s-1 estimate: derived from experimental values [1]
ktranslation 0.167 s-1 lock defined translation rate for Lactonase [1]

Models

CellDesigner (SBML file)

LactonaseProduction CellDesigner.png

Matlab (SBML file)

LactonaseProduction Matlab.jpg

Simulation

During the first 50000 seconds, the input signal TetR (no aTc) is held constant at 5E-5 s-1. This results in a background signal of lactonase which will convert a small part of the HSL into hydroxy acid. From 50000 till 80000 seconds, the input signal is at its maximum value: 0.0125 s-1 (aTc added). This results in an increase of lactonase which converts almost every HSL molecule into the hydroxy acid. The timer is set to 0. After a delay of approximately 20000 seconds, the amount of HSL starts to increase again: the clock is ticking. A shorter pulse of TetR (1000 seconds) only partially resets the timer: not all the HSL is converted into hydroxy acid. The graph has amounts (number of molecules in the cell) plotted vs time, measured in seconds.

Sim lactonaseproduction 1.png

References

Bibliography

[1]“Berkeley2006-RiboregulatorsMain - IGEM”; http://parts2.mit.edu/wiki/index.php/Berkeley2006-RiboregulatorsMain.
 
[2]J.A. Bernstein et al., “Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, Jul. 2002, pp. 9697–9702.
 
[3]J.B. Andersen et al., “New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria,” Applied and Environmental Microbiology, vol. 64, Jun. 1998, pp. 2240–2246.
 
[4]G.M. Skinner et al., “Promoter Binding, Initiation, and Elongation By Bacteriophage T7 RNA Polymerase: A SINGLE-MOLECULE VIEW OF THE TRANSCRIPTION CYCLE,” J. Biol. Chem., vol. 279, Jan. 2004, pp. 3239-3244.
 

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