Talk:Tokyo/Preliminary assays
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Latest revision as of 03:15, 22 October 2007
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AHL preparation
Experimental precision and accurecy
AHL assay
Purpose1:
To practice 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
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
Purpose:
To see how the fluorescence changes though time course.
Samples:
ΔP-GFP in pTrc99A (neg. con.)
placQI in pTrc99A (pos. con.)
Only LB (blank)
Procedure:
Result:
Conclusion:
Wash
Purpose:
To determine the actual fluorescence of GFP expressed under the promoters by removing other influencial facters such as culture media.
Samples:
A4 placQI in pTrc99A (pos. con.)
A4ΔP in pTrc99A
A4 hybrid promoter in pBR322
Procedure:
AHL assay Standard protocol
Wash
Result:
Conclusion:
Detailed AHL assay
Purpose:
To determine the AHL dependent behaviour in narrower range of AHL concentration for the analyses and simulation.
To deternime the Hill coefficient for the relation between the concentration of AHL and our lux^lac hybrid promoter.
Samples:
A4 placQI in pTrc99A (pos. con.)
A4ΔP in pTrc99A (neg. con.)
A4 hybrid promoter in pBR322
Procedure:
AHL assay Standard protocol
Wash
Result:
Conclusion:
New-AHL assay
Purpose:
To determine and compare the activity of the AHL used so far and that of the newly perchased AHL.
Samples:
A4 placQI in pTrc99A (pos. con.)
A4ΔP in pTrc99A (neg. con.)
A4 hybrid promoter in pBR322
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
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. pTrc99A + [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
