Tokyo/Preliminary assays
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Revision as of 16:15, 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:
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:
The old and the newly delivered AHL was used.
AHL assay Standard protocol
The final concantrations of AHL applied here were 0.01, 0.1, 0.5, 1, 10, 100, and 1000 nM.
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
