Team:KULeuven/Model/Reset

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Latest revision as of 14:56, 3 October 2008

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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

ODE's

NOT AVAILABLE

Parameters

*Parameter values (Pulse Generator)*
Name	Value	Comments
Degradation rates
d_{RNA_cI}	0.00462 s^-1
d_cI	7.0E-4 s^-1
d_{RNA_Lac}	0.00231 s^-1
d_Lac	2.888E-4 s^-1
d_{RNA_Ribokey:cI}	0.00231 s^-1
Dissociation constants
K_Ribokey:cI	0.00212	k_ass/k_diss for the Ribokey cI complex
K_cI	0.00337	binding cI on cI-Promotor
Transcription rates
k_{RNA_cI}	0.025 s^-1	maximal transcription rate RNA cI (no cI repressor present)
k_{RNA_Lac}	0.025 s^-1
Translation rates
k_cI	0.167 s^-1
k_Lac	0.167 s^-1	RBS is B0032 (efficiency 0.3)
Hill cooperativity
n_cI	2.0

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

Models

CellDesigner (SBML file)

Matlab

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

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

ODE's

Parameters

*Parameter values Constant Lactonase Production*
Name	Value	Comments	Reference
Degradation rates
d_aiiA	d_LVA = 2.814E-4 s^-1	LVA-tag reduces lifetime to 40 minutes	[3]
d_{closed mRNA aiiA}	0.0046209812 s^-1	estimate: because this RNA isn't translated, it degrades faster	[2]
d_{open mRNA aiiA}	0.0023104906 s^-1		[2]
d_{mRNA aiiA complex}	0.0023104906 s^-1		[2]
T7 Transcription
K_T7	421	dissociation constant, recalculated to remove units	[4]
k_max	0.044 s^-1	maximal T7 transcription rate	[4]
Key-Lock constants
K_{eq 1}	0,015 [M]	between closed and open Lactonase mRNA, modeled for competition, experimental	[1]
K_{eq 2}	0.0212 [M]	between closed Lactonase mRNA and key unlocked mRNA complex, modeled for competition, experimental	[1]
k_dis1	0.00416 s^-1	estimate: derived from experimental values	[1]
k_complex1	0.00237 s^-1	estimate: derived from experimental values	[1]
k_closed	500 s^-1	estimate: derived from experimental values	[1]
k_open	7.5 s^-1	estimate: derived from experimental values	[1]
k_translation	0.167 s^-1	lock defined translation rate for Lactonase	[1]

Models

CellDesigner (SBML file)

Matlab (SBML file)

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.

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.

@@ Line 1: / Line 1: @@
-{{:Team:KULeuven/Tools/Navigation_Bar}}
+{{:Team:KULeuven/Tools/Styling}}
+{{:Team:KULeuven/Tools/Scripting}}
+{{:Team:KULeuven/Tools/Header}}
+[[Image:logo_reset.jpg|120px|right]]
 == 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 ===
@@ Line 10: / Line 18: @@
 ==== ODE's ====
+<html>
+<body>
+<p>
+<img border="0" src="https://2008.igem.org/wiki/skins/common/images/icons/fileicon-pdf.png" width="65" height="60">
+NOT AVAILABLE
+</p>
+</body>
+</html>
 ==== Parameters ====
@@ Line 31: / Line 48: @@
 | 7.0E-4 s<sup>-1</sup>
 |
-| [http://parts.mit.edu/igem07/index.php?title=ETHZ/Parameters link]
+|
 |-
 | d<sub>RNA_Lac</sub>
@@ Line 58: / Line 75: @@
 | 0.00337
 | binding cI on cI-Promotor
-| [http://parts.mit.edu/igem07/index.php?title=ETHZ/Parameters link]
+|
 |-
 ! colspan="4" style="border-bottom: 1px solid #003E81;" | Transcription rates
 |-
 | k<sub>RNA_cI</sub>
-| 0.025? s<sup>-1</sup>
+| 0.025 s<sup>-1</sup>
 | maximal transcription rate RNA cI (no cI repressor present)
 |
 |-
 | k<sub>RNA_Lac</sub>
-| 0.025? s<sup>-1</sup>
+| 0.025 s<sup>-1</sup>
 |
 |
@@ Line 75: / Line 92: @@
 |-
 | k<sub>cI</sub>
-| 0.167? s<sup>-1</sup>
+| 0.167 s<sup>-1</sup>
 |
 |
@@ Line 82: / Line 99: @@
 | 0.167 s<sup>-1</sup>
 | RBS is B0032 (efficiency 0.3)
-| [http://partsregistry.org/Part:BBa_B0032 link]
+|
 |-
 ! colspan="4" style="border-bottom: 1px solid #003E81;" | Hill cooperativity
@@ Line 89: / Line 106: @@
 | 2.0
 |
-| [http://parts.mit.edu/igem07/index.php?title=ETHZ/Parameters link]
+|
 |}
+<br>
+All references for parameters can be found below, in the new section.
 === Models ===
@@ Line 100: / Line 121: @@
 ==== Matlab ====
-The diagram for the pulse generator system is shown below:
 [[Image:Pulse_Generator_Matlab.jpg|center]]
-Following figure shows the repons of the pulse generator to a step signal of T7 polymerase.
+=== 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 ==
+<div style="padding-left: 2em; float: right">[[Image:pictogram_lactonaseproduction.png|120px]]</div>
+=== Position in the system ===
+The Constant Lactonase Production-system is directly linked to the [[Team:KULeuven/Model/Filter | 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: [https://2008.igem.org/Team:KULeuven/Project/Reset Project:Reset]
+[[Image:Modeling Reset.PNG|center]]
+==== ODE's ====
+<html>
+<body>
+<p>
+<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">
+</a>
+</p>
+</body>
+</html>
+==== Parameters ====
+{| width=80% style="border: 1px solid #003E81; background-color: #EEFFFF;"
+|+ ''Parameter values Constant Lactonase Production''
+! width=15% | Name
+! width=15% | Value
+! width=40% | Comments
+! width=10% | Reference
+|-
+! colspan="4" style="border-bottom: 1px solid #003E81;" | Degradation rates
+|-
+| d<sub>aiiA</sub>
+| | d<sub>LVA</sub> = 2.814E-4 s<sup>-1</sup>
+| LVA-tag reduces lifetime to 40 minutes
+| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [3<html>]</html>]
+|-
+| d<sub>closed mRNA aiiA</sub>
+| 0.0046209812 s<sup>-1</sup>
+| estimate: because this RNA isn't translated, it degrades faster
+| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [2<html>]</html>]
+|-
+| d<sub>open mRNA aiiA</sub>
+| 0.0023104906 s<sup>-1</sup>
+|
+| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [2<html>]</html>]
+|-
+| d<sub>mRNA aiiA complex</sub>
+| 0.0023104906 s<sup>-1</sup>
+|
+| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [2<html>]</html>]
+|-
+! colspan="4" style="border-bottom: 1px solid #003E81;" | T7 Transcription
+|-
+| K<sub>T7</sub>
+| 421
+| dissociation constant, recalculated to remove units
+| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [4<html>]</html>]
+|-
+| k<sub>max</sub>
+| 0.044 s<sup>-1</sup>
+| maximal T7 transcription rate
+| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [4<html>]</html>]
+|-
+! colspan="4" style="border-bottom: 1px solid #003E81;" | Key-Lock constants
+|-
+| K<sub>eq 1</sub>
+| 0,015 [M]
+| between closed and open Lactonase mRNA, modeled for competition, experimental
+| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [1<html>]</html>]
+|-
+| K<sub>eq 2</sub>
+| 0.0212 [M]
+| between closed Lactonase mRNA and key unlocked mRNA complex, modeled for competition, experimental
+| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [1<html>]</html>]
+|-
+| k<sub>dis1</sub>
+| 0.00416 s<sup>-1</sup>
+| estimate: derived from experimental values
+| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [1<html>]</html>]
+|-
+| k<sub>complex1</sub>
+| 0.00237 s<sup>-1</sup>
+| estimate: derived from experimental values
+| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [1<html>]</html>]
+|-
+| k<sub>closed</sub>
+| 500 s<sup>-1</sup>
+| estimate: derived from experimental values
+| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [1<html>]</html>]
+|-
+| k<sub>open</sub>
+| 7.5 s<sup>-1</sup>
+| estimate: derived from experimental values
+| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [1<html>]</html>]
+|-
+| k<sub>translation</sub>
+| 0.167 s<sup>-1</sup>
+| lock defined translation rate for Lactonase
+| [https://2008.igem.org/Team:KULeuven/Model/Reset#References [1<html>]</html>]
+|}
+=== Models ===
+==== CellDesigner ([https://static.igem.org/mediawiki/2008/0/02/LactonaseProduction_CellDesigner.zip SBML file]) ====
+[[Image:LactonaseProduction_CellDesigner.png|500px|center]]
+==== Matlab ([https://static.igem.org/mediawiki/2008/8/8d/LactonaseProduction_Matlab.zip SBML file]) ====
+[[Image:LactonaseProduction_Matlab.jpg|500px|center]]
+=== Simulation ===
+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.
+[[Image:Sim_lactonaseproduction_1.png|700px|center]]
+=== References ===
+<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en">
+<head>
+<meta http-equiv="Content-Type" content="text/html; charset=utf-8"/>
+<title>Bibliography</title>
+</head>
+<body>
+<table style="border-collapse:collapse;line-height:1.1em;">
+<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>
+</html>

Team:KULeuven/Model/Reset

From 2008.igem.org

Latest revision as of 14:56, 3 October 2008

Contents

Pulse Generator

Position in the system

Describing the system

ODE's

Parameters

Models

CellDesigner (SBML file)

Matlab

Problem

Constant Lactonase Production

Position in the system

Describing the system

ODE's

Parameters

Models

CellDesigner (SBML file)

Matlab (SBML file)

Simulation

References