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Team:KULeuven/14 July 2008
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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 | ||
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| - | + | '''OUTPUT:''' | |
* Compare GFP-LVA with normal GFP. | * Compare GFP-LVA with normal GFP. | ||
* You will need input to switch production of reporter on/off. | * You will need input to switch production of reporter on/off. | ||
| - | + | '''MEMORY:''' | |
* Until 2nd terminator set, change c2 P22 to GFP. | * Until 2nd terminator set, change c2 P22 to GFP. | ||
* anti-LuxI --> quantitate with Q-RT-PCR (compare with LuxI expression INVERTER). | * anti-LuxI --> quantitate with Q-RT-PCR (compare with LuxI expression INVERTER). | ||
| - | T7 | + | '''T7 (with LVA tag):''' |
* Either with Antibodies (Western blot of ...), or | * Either with Antibodies (Western blot of ...), or | ||
* Build ''p(T7)-GFP''. | * Build ''p(T7)-GFP''. | ||
| - | + | '''FILTER:''' | |
* Build ''filter + p(T7)-lock3d-GFP''. | * Build ''filter + p(T7)-lock3d-GFP''. | ||
* You will need input. | * You will need input. | ||
| Line 35: | Line 36: | ||
| - | + | '''INVERTER:''' | |
* Build inverter. | * Build inverter. | ||
* You will need input + filter. | * You will need input + filter. | ||
| Line 41: | Line 42: | ||
| - | + | '''PULSE GENERATOR:''' | |
* Replace aiiA --> GFP. | * Replace aiiA --> GFP. | ||
| - | + | '''CELL DEATH:''' | |
* Entire system with YFP,GFP,... instead of ccdB. | * Entire system with YFP,GFP,... instead of ccdB. | ||
Latest revision as of 18:05, 6 October 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 (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.
- ?
