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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== AHL assay==
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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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===Date: ===
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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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2007/09/06, 07
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===Purpose1: ===
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To practive AHL assay.
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===Purpose2: ===
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To check if AHL works. 
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===Samples: ===
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== ==
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<br>luxR + GFP in DH5a (AHL-activated promoter)
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'''Preliminary assays were conducted'''
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<br>placQI + GFP in DH5a (pos. con.)
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<br>- '''to practice and experience the assays with AHL and IPTG'''
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<br>pSBΔP+ GFP (neg. con.)
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<br>- '''to check if and how our materials actually worked.'''
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<br>ΔP = promoter deleted
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== [[Tokyo/AHL preparation |AHL preparation]] ==
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===Procedure: ===
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== [[Tokyo/Experimental precision and accurecy|Experimental precision and accuracy]] ==
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<br>prepare overnight culture (3 tubes for luxR, 2 tubes for each controls)
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== [[Tokyo/AHL assay practice|AHL assay practice]] ==
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<br>take 30 ul of the overnight culture into LB + Kan (⇒fresh culture)
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== [[Tokyo/Lux-lac hybrid promoter activation check|Lux-lac hybrid promoter activation check]] ==
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<br>incubate the fresh culture until the observed OD reaches around 0.80.
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== [[Tokyo/OD correction|OD correction]] ==
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<br>prepare and add AHL mixture; final concentration of AHL = 6 pM
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== [[Tokyo/Wash|Wash]] ==
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<br>incubation for 2 hours
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== [[Tokyo/New AHL check|New AHL check]] ==
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<br>1st FLA measurement for GFP expression
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<!--== [[Tokyo/Hybrid promoter activation & repression check|Hybrid promoter activation & repression check ]] ==
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<br>2nd FLA measurement for GFP expression
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<br>add the same amout AHL as the first addition
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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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===Date: ===
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2007/09/15
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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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Lux-lac hybrid promoter (before blue light check)
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Lux-lac hybrid promoter (after blue light check)
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placQI (pos. con.)
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modified A4 (promoter deleted) (neg. con.)
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===Procedure: ===
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===Result: ===
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===Conclusion: ===
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== OD correction ==
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===Date: ===
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2007/09/21
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===Purpose: ===
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===Samples: ===
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===Procedure: ===
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===Result: ===
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===Conclusion: ===
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== Wash ==
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===Date: ===
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20070925
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===Purpose: ===
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===Samples: ===
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===Procedure: ===
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===Result: ===
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===Conclusion: ===
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== Detailed AHL assay ==
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===Date: ===
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20070925
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===Purpose: ===
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===Samples: ===
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===Procedure: ===
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===Result: ===
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===Conclusion: ===
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== New-AHL assay ==
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===Date: ===
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20070926
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===Purpose: ===
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===Samples: ===
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===Procedure: ===
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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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=== Date: ===
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2007/09/20, 21
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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. pTrc322 TetR + [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>
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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