From 2007.igem.org

Contents 1 AHL preparation 2 Experimental precision and accurecy 3 AHL assay 3.1 Purpose1: 3.2 Purpose2: 3.3 Samples: 3.4 Procedure: 3.5 Result: 3.6 Conclusion: 4 AHL assay 4.1 Purpose: 4.2 Samples: 4.3 Procedure: 4.4 Result: 4.5 Conclusion: 5 OD correction 5.1 Purpose: 5.2 Samples: 5.3 Procedure: 5.4 Result: 5.5 Conclusion: 6 Wash 6.1 Purpose: 6.2 Samples: 6.3 Procedure: 6.4 Result: 6.5 Conclusion: 7 Detailed AHL assay 7.1 Purpose: 7.2 Samples: 7.3 Procedure: 7.4 Result: 7.5 Conclusion: 8 New-AHL assay 8.1 Purpose: 8.2 Samples: 8.3 Procedure: 8.4 Result: 8.5 Conclusion: 9 Lux-lac hybrid promoter Check 9.1 Purpose 1: 9.2 Purpose 2: 9.3 Samples: 9.4 Procedure:

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

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