Team:KULeuven/14 July 2008

From 2008.igem.org

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==PRactical==
We will meet at 11 o'clock in room 00.210
We will meet at 11 o'clock in room 00.210
-
== Testing the system ==
+
==results==
 +
=== Testing the system ===
 +
 
 +
'''INPUT:'''
 +
* In order to use INPUT, use/test part 1 of 'Cell Death' (luxR, TetR under constitutive promotor).
 +
* Test input-output as such (= test OmpR promotor etc), i.e. test the input with the output.
 +
 
 +
 
 +
Output:
 +
* Compare GFP-LVA with normal GFP.
 +
* You will need input to switch production of reporter on/off.
 +
 
 +
 
 +
Memory:
 +
* Until 2nd terminator set, change c2 P22 to GFP.
 +
* anti-LuxI --> quantitate with Q-RT-PCR (compare with LuxI expression INVERTER).
 +
 
 +
 
 +
T7 characterization (with LVA tag):
 +
* Either with Antibodies (Western blot of ...), or
 +
* Build ''p(T7)-GFP''.
 +
 
 +
 
 +
Filter:
 +
* Build ''filter + p(T7)-lock3d-GFP''.
 +
* You will need input.
 +
* Characterize filter with short pulses (input).
 +
 
 +
 
 +
Inverter:
 +
* Build inverter.
 +
* You will need input + filter.
 +
* Q-RT-PCR of LuxI.
 +
 
 +
 
 +
Pulse generator:
 +
* Replace aiiA --> GFP.
 +
 
-
voor gebruik INPUT, gebruik/test deel 1 van Cell Death Part (luxR, TetR onder constit promotor)
+
Cell Death:
-
# Test input output as such (= test OmpR promotor etc)
+
* Entire system with YFP,GFP,... instead of ccdB.
-
# MEMORY TEST
+
-
## tot aan 2de terminator set, change c2 P22 to GFP
+
-
## anti-LuxI --> quantitate with Q-RT-PCR (vergelijk met LuxI expressie INVERTER)
+
-
# T7 karakterisatie (met LVA tag)
+
-
## oftewel met Ab's (Western blot of ...), p(T7)-GFP
+
-
# FILTER TEST = filter + p(T7)-lock3d-GFP
+
-
# INVERTER TEST: Q-RT-PCR LuxI
+
-
# PULSE GENERATOR TEST: aiiA --> GFP
+
-
# CELL DEATH: Entire system with YFP ofzo ipv ccdB
+
 +
=== Modeling ===
-
input <br>
+
We studied the (sub)components of CellDesigner.
-
test met output <br>
+
-
hang filter achter input <br>
+
-
:# karakterisatie filter via korte pulsen
+
-
:# test pulse generator
+
-
maak inverter <br>
+
-
maak memory <br>
+
-
hang deel 2 cell death (ccdB) achter deel 1 <br>
+
-
== Modeling ==
+
'''Plugin SBML Squeezer''' was installed to automatically generate ODEs.
-
# Herbekijken deelcomponenten in CellDesigner
+
We started our quest for kinetic parameters (we made a collection at our [https://2008.igem.org/Team:KULeuven/Model/KineticConstants Modeling] page):
-
# Plugin SBML Squeezer geinstalleerd voor automatische generatie ODE stelsels
+
* Degradation of T7 with LVA tag was obtained by the interpolation of the influence of LVA on the degradation of GFP. We presume that this is valuable for every protein, including T7 polymerase.
-
# Zoektocht gestart naar kinetische parameters (verzameling aangelegd in [[Team:KULeuven/Modeling]] page
+
* ?
-
## Degradatie voor pT7 met LVA tag bekomen uit interpolatie LVA invloed op GFP afbraak, aangenomen dat dit geldig is voor eender welk proteine, en dus eveneens voor T7 polymerase
+
-
## ?
+

Revision as of 21:00, 16 September 2008

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Contents

PRactical

We will meet at 11 o'clock in room 00.210

results

Testing the system

INPUT:

  • In order to use INPUT, use/test part 1 of 'Cell Death' (luxR, TetR under constitutive promotor).
  • Test input-output as such (= test OmpR promotor etc), i.e. test the input with the output.


Output:

  • Compare GFP-LVA with normal GFP.
  • You will need input to switch production of reporter on/off.


Memory:

  • Until 2nd terminator set, change c2 P22 to GFP.
  • anti-LuxI --> quantitate with Q-RT-PCR (compare with LuxI expression INVERTER).


T7 characterization (with LVA tag):

  • Either with Antibodies (Western blot of ...), or
  • Build p(T7)-GFP.


Filter:

  • Build filter + p(T7)-lock3d-GFP.
  • You will need input.
  • Characterize filter with short pulses (input).


Inverter:

  • Build inverter.
  • You will need input + filter.
  • Q-RT-PCR of LuxI.


Pulse generator:

  • Replace aiiA --> GFP.


Cell Death:

  • Entire system with YFP,GFP,... instead of ccdB.


Modeling

We studied the (sub)components of CellDesigner.

Plugin SBML Squeezer was installed to automatically generate ODEs.

We started our quest for kinetic parameters (we made a collection at our Modeling page):

  • Degradation of T7 with LVA tag was obtained by the interpolation of the influence of LVA on the degradation of GFP. We presume that this is valuable for every protein, including T7 polymerase.
  •  ?
Retrieved from "http://2008.igem.org/Team:KULeuven/14_July_2008"