Tokyo/Preliminary assays
From 2007.igem.org
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===Purpose: === | ===Purpose: === | ||
| + | To determine the actual fluorescence of GFP expressed under the promoters by removing other influencial facters such as culture media. | ||
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===Samples: === | ===Samples: === | ||
===Procedure: === | ===Procedure: === | ||
Revision as of 15:48, 20 October 2007
Contents |
AHL preparation
AHL assay
Date:
2007/09/06, 07
Purpose1:
To practive AHL assay.
Purpose2:
To check if AHL works.
Samples:
luxR + GFP in DH5a (AHL-activated promoter)
placQI + GFP in DH5a (pos. con.)
pSBΔP+ GFP (neg. con.)
ΔP = promoter deleted
Procedure:
The final concentration of AHL is 6 pM.
Result:
Conclusion:
6pM of AHL is not enough to activate the AHL-activated luxR promoter.
AHL assay
Date:
2007/09/15
Purpose:
check if lux-lac hybrid promoter works
Samples:
Lux-lac hybrid promoter (before blue light check)
Lux-lac hybrid promoter (after blue light check)
placQI (pos. con.)
modified A4 (promoter deleted) (neg. con.)
Procedure:
AHL assay Standard protocol
The fluorescence of GFP was measured every 1 hour after AHL was added.
Result:
Conclusion:
OD correction
Date:
2007/09/21
Purpose:
Samples:
ΔP-GFP in pTrc99A (neg. con.)
placQI in pTrc99A (pos. con.)
Only LB (blank)
Procedure:
Result:
Conclusion:
Wash
Date:
20070925
Purpose:
To determine the actual fluorescence of GFP expressed under the promoters by removing other influencial facters such as culture media.
Samples:
Procedure:
AHL assay Standard protocol
Wash
Result:
Conclusion:
Detailed AHL assay
Date:
20070925
Purpose:
Samples:
Procedure:
Result:
Conclusion:
New-AHL assay
Date:
20070926
Purpose:
Samples:
Procedure:
Result:
Conclusion:
Lux-lac hybrid promoter Check
Date:
2007/09/20, 21
Purpose 1:
To check if LacI hybrid promoter is activated by AHL and repressed by LacI.
Purpose 2:
To obtain parameters of LacI hybrid promoter for computational simulation.
Samples:
1. pTrc99A + [LacI hybrid promoter – GFP] (LacI+)
2. pBR322 TetR + [LacI hybrid promoter – GFP] (lacI-)
3. pTrc99A + [placQI – GFP] (placQI is constitutive promoter)
4. pTrc322 TetR + [LacI promoter – GFP]
5. pBR322 TetR + [LacI promoter – GFP]
Repressor LacI expression:
pTrc99A expresses LacI
pBR322 TetR does NOT express LacI
Antibiotics resistance:
pTrc99A gives ampicillin-resistance
pBR322 TetR gives ampicillin-resistance
[LacI hybrid promoter – GFP] gives kanamicin-resistance
[placQI – GFP] gives kanamicin-resistance
