Talk:Tokyo/Preliminary assays

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(Difference between revisions)
 
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== [[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 ]]

Latest revision as of 03:15, 22 October 2007

Contents

AHL preparation

Experimental precision and accurecy

AHL assay

Purpose1:

To practice 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:


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:


Lux-lac hybrid promoter (before blue light check)
Lux-lac hybrid promoter (after blue light check)
placQI (pos. con.)
modified A4 (promoter deleted) (neg. con.)

Procedure:

AHL assay Standard protocol

Result:

Conclusion:

OD correction

Purpose:


To see how the fluorescence changes though time course.

Samples:


ΔP-GFP in pTrc99A (neg. con.)
placQI in pTrc99A (pos. con.)
Only LB (blank)

Procedure:

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:


A4 placQI in pTrc99A (pos. con.)
A4ΔP in pTrc99A
A4 hybrid promoter in pBR322

Procedure:

AHL assay Standard protocol
Wash

Result:

Conclusion:

Detailed AHL assay

Purpose:


To determine the AHL dependent behaviour in narrower range of AHL concentration for the analyses and simulation.
To deternime the Hill coefficient for the relation between the concentration of AHL and our lux^lac hybrid promoter.

Samples:


A4 placQI in pTrc99A (pos. con.)
A4ΔP in pTrc99A (neg. con.)
A4 hybrid promoter in pBR322

Procedure:


AHL assay Standard protocol
Wash

Result:

Conclusion:

New-AHL assay

Purpose:


To determine and compare the activity of the AHL used so far and that of the newly perchased AHL.

Samples:


A4 placQI in pTrc99A (pos. con.)
A4ΔP in pTrc99A (neg. con.)
A4 hybrid promoter in pBR322

Procedure:


The old and the newly delivered AHL was used.
AHL assay Standard protocol
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

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:


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


AHL assay Standard protocol

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