Tokyo/Preliminary assays

From 2007.igem.org

(Difference between revisions)
(Purpose:)
 
(62 intermediate revisions not shown)
Line 1: Line 1:
-
== [[Tokyo/AHL preparation |AHL preparation]] ==
+
__NOTOC__
-
== AHL assay==
+
<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]]
-
+
<br><br>
-
===Date: ===
+
[[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]]
-
2007/09/06, 07
+
-
===Purpose1: ===
+
-
To practive AHL assay.
+
-
===Purpose2: ===
+
-
To check if AHL works. 
+
-
===Samples: ===
+
== ==
-
<br>luxR + GFP in DH5a (AHL-activated promoter)
+
'''Preliminary assays were conducted'''
-
<br>placQI + GFP in DH5a (pos. con.)
+
<br>- '''to practice and experience the assays with AHL and IPTG'''
-
<br>pSBΔP+ GFP (neg. con.)
+
<br>- '''to check if and how our materials actually worked.'''
-
<br>ΔP = promoter deleted
+
== [[Tokyo/AHL preparation |AHL preparation]] ==
-
===Procedure: ===
+
== [[Tokyo/Experimental precision and accurecy|Experimental precision and accuracy]] ==
-
<br>prepare overnight culture (3 tubes for luxR, 2 tubes for each controls)
+
== [[Tokyo/AHL assay practice|AHL assay practice]] ==
-
<br>take 30 ul of the overnight culture into LB + Kan (⇒fresh culture)
+
== [[Tokyo/Lux-lac hybrid promoter activation check|Lux-lac hybrid promoter activation check]] ==
-
<br>incubate the fresh culture until the observed OD reaches around 0.80.
+
== [[Tokyo/OD correction|OD correction]] ==
-
<br>prepare and add AHL mixture; final concentration of AHL = 6 pM
+
== [[Tokyo/Wash|Wash]] ==
-
<br>incubation for 2 hours
+
== [[Tokyo/New AHL check|New AHL check]] ==
-
<br>1st FLA measurement for GFP expression
+
<!--== [[Tokyo/Hybrid promoter activation & repression check|Hybrid promoter activation & repression check ]] ==
-
<br>2nd FLA measurement for GFP expression
+
-
<br>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 ==
+
-
 
+
-
[[Image:luxlachybrid.jpg]]
+
-
 
+
-
=== 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: ===
+
-
<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. pTrc322 TetR + [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>
 +
-->

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