Tokyo/Preliminary assays

From 2007.igem.org

(Difference between revisions)
Line 1: Line 1:
== [[Tokyo/AHL preparation |AHL preparation]] ==
== [[Tokyo/AHL preparation |AHL preparation]] ==
 +
== AHL assay==
 +
 +
===Date: ===
 +
2007/09/06, 07
 +
===Purpose1: ===
 +
To practive 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>prepare overnight culture (3 tubes for luxR, 2 tubes for each controls)
 +
<br>take 30 ul of the overnight culture into LB + Kan (⇒fresh culture)
 +
<br>incubate the fresh culture until the observed OD reaches around 0.80.
 +
<br>prepare and add AHL mixture; final concentration of AHL = 6 pM
 +
<br>incubation for 2 hours
 +
<br>1st FLA measurement for GFP expression
 +
<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.
== OD correction ==
== OD correction ==

Revision as of 13:27, 20 October 2007

Contents

AHL preparation

AHL assay

Date:

2007/09/06, 07

Purpose1:

To practive 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:


prepare overnight culture (3 tubes for luxR, 2 tubes for each controls)
take 30 ul of the overnight culture into LB + Kan (⇒fresh culture)
incubate the fresh culture until the observed OD reaches around 0.80.
prepare and add AHL mixture; final concentration of AHL = 6 pM
incubation for 2 hours
1st FLA measurement for GFP expression
2nd FLA measurement for GFP expression
add the same amout AHL as the first addition

Result:

Conclusion:

6pM of AHL is not enough to activate the AHL-activated luxR promoter.

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

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:


1. pTrc99A + [LacI hybrid promoter – GFP] (LacI+)
2. pBR322 TetR + [LacI hybrid promoter – GFP] (lacI-)
3. pTrc99A + [placQI – GFP] (placQI is constitutive promoter)
4. pTrc322 TetR + [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

Procedure: