|
|
| Line 1: |
Line 1: |
| - | == [[Tokyo/AHL preparation |AHL preparation]] ==
| |
| - | == [[Tokyo/Experimental precision and accurecy|Experimental precision and accurecy]] ==
| |
| | | | |
| - | == AHL assay==
| |
| - |
| |
| - |
| |
| - |
| |
| - | ===Purpose1: ===
| |
| - | To practice AHL assay.
| |
| - |
| |
| - | ===Purpose2: ===
| |
| - | To check if AHL works.
| |
| - |
| |
| - | ===Samples: ===
| |
| - | <br>luxR + GFP in DH5a (AHL-activated promoter)
| |
| - | <br>placQI + GFP in DH5a (pos. con.)
| |
| - | <br>pSBΔP+ GFP (neg. con.)
| |
| - |
| |
| - | <br>ΔP = promoter deleted
| |
| - |
| |
| - | ===Procedure: ===
| |
| - | <br>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: ===
| |
| - | <br>Lux-lac hybrid promoter (before blue light check)
| |
| - | <br>Lux-lac hybrid promoter (after blue light check)
| |
| - | <br>placQI (pos. con.)
| |
| - | <br>modified A4 (promoter deleted) (neg. con.)
| |
| - |
| |
| - | ===Procedure: ===
| |
| - | [[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
| |
| - |
| |
| - | ===Result: ===
| |
| - |
| |
| - | ===Conclusion: ===
| |
| - |
| |
| - | == OD correction ==
| |
| - |
| |
| - |
| |
| - |
| |
| - | ===Purpose: ===
| |
| - | <br>To see how the fluorescence changes though time course.
| |
| - |
| |
| - | ===Samples: ===
| |
| - | <br>ΔP-GFP in pTrc99A (neg. con.)
| |
| - | <br>placQI in pTrc99A (pos. con.)
| |
| - | <br>Only LB (blank)
| |
| - |
| |
| - | ===Procedure: ===
| |
| - | [[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
| |
| - |
| |
| - | ===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: ===
| |
| - | <br>A4 placQI in pTrc99A (pos. con.)
| |
| - | <br>A4ΔP in pTrc99A
| |
| - | <br>A4 hybrid promoter in pBR322
| |
| - |
| |
| - | ===Procedure: ===
| |
| - | [[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
| |
| - | <br>[[Tokyo/Wash |Wash ]]
| |
| - |
| |
| - | ===Result: ===
| |
| - | ===Conclusion: ===
| |
| - |
| |
| - |
| |
| - | == Detailed AHL assay ==
| |
| - |
| |
| - |
| |
| - |
| |
| - | ===Purpose: ===
| |
| - | <br>To determine the AHL dependent behaviour in narrower range of AHL concentration for the analyses and simulation.
| |
| - | <br>To deternime the Hill coefficient for the relation between the concentration of AHL and our lux^lac hybrid promoter.
| |
| - |
| |
| - | ===Samples: ===
| |
| - | <br>A4 placQI in pTrc99A (pos. con.)
| |
| - | <br>A4ΔP in pTrc99A (neg. con.)
| |
| - | <br>A4 hybrid promoter in pBR322
| |
| - |
| |
| - | ===Procedure: ===
| |
| - | <br>[[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
| |
| - | <br>[[Tokyo/Wash |Wash ]]
| |
| - |
| |
| - | ===Result: ===
| |
| - | ===Conclusion: ===
| |
| - |
| |
| - | == New-AHL assay ==
| |
| - |
| |
| - |
| |
| - |
| |
| - | ===Purpose: ===
| |
| - | <br>To determine and compare the activity of the AHL used so far and that of the newly perchased AHL.
| |
| - |
| |
| - | ===Samples: ===
| |
| - | <br>A4 placQI in pTrc99A (pos. con.)
| |
| - | <br>A4ΔP in pTrc99A (neg. con.)
| |
| - | <br>A4 hybrid promoter in pBR322
| |
| - |
| |
| - | ===Procedure: ===
| |
| - | <br>The old and the newly delivered AHL was used.
| |
| - | <br>[[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
| |
| - | <br>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 ==
| |
| - |
| |
| - | [[Image:luxlachybrid.jpg]]
| |
| - |
| |
| - |
| |
| - |
| |
| - | ===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: ===
| |
| - | <br>1. pTrc99A + [LacI hybrid promoter – GFP] (LacI+)
| |
| - | <br>2. pBR322 TetR + [LacI hybrid promoter – GFP] (lacI-)
| |
| - | <br>3. pTrc99A + [placQI – GFP] (placQI is constitutive promoter)
| |
| - | <br>4. pTrc99A + [LacI promoter – GFP]
| |
| - | <br>5. pBR322 TetR + [LacI promoter – GFP]
| |
| - |
| |
| - | <br>'''Repressor LacI expression:'''
| |
| - | <br>pTrc99A expresses LacI
| |
| - | <br>pBR322 TetR does NOT express LacI
| |
| - |
| |
| - | <br>'''Antibiotics resistance:'''
| |
| - | <br>pTrc99A gives ampicillin-resistance
| |
| - | <br>pBR322 TetR gives ampicillin-resistance
| |
| - | <br>[LacI hybrid promoter – GFP] gives kanamicin-resistance
| |
| - | <br>[placQI – GFP] gives kanamicin-resistance
| |
| - |
| |
| - | ===Procedure:===
| |
| - | <br>[[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
| |
