Tokyo/Preliminary assays
From 2007.igem.org
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:
prepare overnight culture (3 tubes for luxR, 2 tubes for each controls)
take 30 ul of the overnight culture into LB + Kan (⇒fresh culture)
incubate the fresh culture until the observed OD reaches around 0.80.
prepare and add AHL mixture; final concentration of AHL = 6 pM
incubation for 2 hours
1st FLA measurement for GFP expression
2nd FLA measurement for GFP expression
add the same amout AHL as the first addition
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:
Procedure:
Result:
Conclusion:
OD correction
Date:
2007/09/21
Purpose:
Samples:
Procedure:
Result:
Conclusion:
Wash
Date:
20070925
Purpose:
Samples:
Procedure:
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
