Tokyo/Preliminary assays

From 2007.igem.org

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== [[Tokyo/AHL preparation |AHL preparation]] ==
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__NOTOC__
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== [[Tokyo/Experimental precision and accurecy|Experimental precision and accurecy]] ==
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<br>[[Tokyo/Works|Works top]]&nbsp;&nbsp;&nbsp;0.[[Tokyo/Works/Hybrid promoter|Hybrid promoter]]&nbsp;&nbsp;&nbsp;1.[[Tokyo/Works/Formulation |Formulation]]　　'''2.[[Tokyo/Works/Assay |Assay1]]'''&nbsp;&nbsp;&nbsp;3.[[Tokyo/Works/Simulation |Simulation]]&nbsp;&nbsp;&nbsp;4.[[Tokyo/Works/Assay2 |Assay2]]&nbsp;&nbsp;&nbsp;5.[[Tokyo/Works/Future works |Future works]]
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<br><br>
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[[Tokyo/Hill function fitting |Objective of this assay]]&nbsp;&nbsp;&nbsp;[[Tokyo/AHL assay|Effect of AHL]]&nbsp;&nbsp;&nbsp;[[Tokyo/IPTG assay|Effect of LacI-IPTG]]　　[[Tokyo/Preliminary assays |Preliminary assays]]
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== AHL assay==
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== ==
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'''Preliminary assays were conducted'''
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<br>- '''to practice and experience the assays with AHL and IPTG'''
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<br>- '''to check if and how our materials actually worked.'''
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== [[Tokyo/AHL preparation |AHL preparation]] ==
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== [[Tokyo/Experimental precision and accurecy|Experimental precision and accuracy]] ==
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== [[Tokyo/AHL assay practice|AHL assay practice]] ==
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== [[Tokyo/Lux-lac hybrid promoter activation check|Lux-lac hybrid promoter activation check]] ==
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== [[Tokyo/OD correction|OD correction]] ==
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== [[Tokyo/Wash|Wash]] ==
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== [[Tokyo/New AHL check|New AHL check]] ==
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<!--== [[Tokyo/Hybrid promoter activation & repression check|Hybrid promoter activation & repression check ]] ==
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===Purpose1: ===
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<br>
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To practive AHL assay.
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-->
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===Purpose2: ===
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To check if AHL works. 
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===Samples: ===
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<br>luxR + GFP in DH5a (AHL-activated promoter)
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<br>placQI + GFP in DH5a (pos. con.)
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<br>pSBΔP+ GFP (neg. con.)
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<br>ΔP = promoter deleted
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===Procedure: ===
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<br>The final concentration of AHL is 6 pM.
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===Result: ===
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===Conclusion: ===
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6pM of AHL is not enough to activate the AHL-activated luxR promoter.
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== AHL assay==
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===Purpose: ===
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check if lux-lac hybrid promoter works
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===Samples: ===
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<br>Lux-lac hybrid promoter (before blue light check)
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<br>Lux-lac hybrid promoter (after blue light check)
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<br>placQI (pos. con.)
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<br>modified A4 (promoter deleted) (neg. con.)
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===Procedure: ===
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[[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
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===Result: ===
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===Conclusion: ===
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== OD correction ==
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===Purpose: ===
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<br>To see how the fluorescence changes though time course.
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===Samples: ===
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<br>ΔP-GFP in pTrc99A (neg. con.)
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<br>placQI in pTrc99A (pos. con.)
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<br>Only LB (blank)
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===Procedure: ===
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[[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
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===Result: ===
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===Conclusion: ===
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== Wash ==
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===Purpose: ===
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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: ===
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<br>A4 placQI in pTrc99A (pos. con.)
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<br>A4ΔP in pTrc99A
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<br>A4 hybrid promoter in pBR322
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===Procedure: ===
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[[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
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<br>[[Tokyo/Wash |Wash ]]
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===Result: ===
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===Conclusion: ===
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== Detailed AHL assay ==
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===Purpose: ===
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<br>To determine the AHL dependent behaviour in narrower range of AHL concentration for the analyses and simulation.
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<br>To deternime the Hill coefficient for the relation between the concentration of AHL and our lux^lac hybrid promoter.
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===Samples: ===
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<br>A4 placQI in pTrc99A (pos. con.)
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<br>A4ΔP in pTrc99A (neg. con.)
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<br>A4 hybrid promoter in pBR322
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===Procedure: ===
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<br>[[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
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<br>[[Tokyo/Wash |Wash ]]
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===Result: ===
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===Conclusion: ===
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== New-AHL assay ==
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===Purpose: ===
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<br>To determine and compare the activity of the AHL used so far and that of the newly perchased AHL.
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===Samples: ===
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<br>A4 placQI in pTrc99A (pos. con.)
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<br>A4ΔP in pTrc99A (neg. con.)
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<br>A4 hybrid promoter in pBR322
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===Procedure: ===
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<br>The old and the newly delivered AHL was used.
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<br>[[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
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<br>The final concantrations of AHL applied here were 0.01, 0.1, 0.5, 1, 10, 100, and 1000 nM.
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===Result: ===
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===Conclusion: ===
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== Lux-lac hybrid promoter Check ==
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[[Image:luxlachybrid.jpg]]
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===Purpose 1:===
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To check if LacI hybrid promoter is activated by AHL and repressed by LacI.
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===Purpose 2:===
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To obtain parameters of LacI hybrid promoter for computational simulation.
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===Samples: ===
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<br>1. pTrc99A + [LacI hybrid promoter – GFP] (LacI+)
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<br>2. pBR322 TetR + [LacI hybrid promoter – GFP] (lacI-)
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<br>3. pTrc99A + [placQI – GFP] (placQI is constitutive promoter)
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<br>4. pTrc99A + [LacI promoter – GFP]
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<br>5. pBR322 TetR + [LacI promoter – GFP]
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<br>'''Repressor LacI expression:'''
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<br>pTrc99A expresses LacI
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<br>pBR322 TetR does NOT express LacI
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<br>'''Antibiotics resistance:'''
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<br>pTrc99A gives ampicillin-resistance
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<br>pBR322 TetR gives ampicillin-resistance
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<br>[LacI hybrid promoter – GFP] gives kanamicin-resistance
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<br>[placQI – GFP] gives kanamicin-resistance
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===Procedure:===
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<br>[[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
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Latest revision as of 05:12, 27 October 2007


Works top   0.Hybrid promoter   1.Formulation　　2.Assay1   3.Simulation   4.Assay2   5.Future works

Objective of this assay   Effect of AHL   Effect of LacI-IPTG　　Preliminary assays

Preliminary assays were conducted
- to practice and experience the assays with AHL and IPTG
- to check if and how our materials actually worked.

AHL preparation

Experimental precision and accuracy

AHL assay practice

Lux-lac hybrid promoter activation check

OD correction

Wash

New AHL check

Retrieved from "http://2007.igem.org/wiki/index.php/Tokyo/Preliminary_assays"