From 2007.igem.org

Contents 1 AHL preparation 2 AHL assay 2.1 Date: 2.2 Purpose1: 2.3 Purpose2: 2.4 Samples: 2.5 Procedure: 2.6 Result: 2.7 Conclusion: 3 AHL assay 3.1 Date: 3.2 Purpose: 3.3 Samples: 3.4 Procedure: 3.5 Result: 3.6 Conclusion: 4 OD correction 4.1 Date: 4.2 Purpose: 4.3 Samples: 4.4 Procedure: 4.5 Result: 4.6 Conclusion: 5 Wash 5.1 Date: 5.2 Purpose: 5.3 Samples: 5.4 Procedure: 5.5 Result: 5.6 Conclusion: 6 Detailed AHL assay 6.1 Date: 6.2 Purpose: 6.3 Samples: 6.4 Procedure: 6.5 Result: 6.6 Conclusion: 7 New-AHL assay 7.1 Date: 7.2 Purpose: 7.3 Samples: 7.4 Procedure: 7.5 Result: 7.6 Conclusion: 8 Lux-lac hybrid promoter Check 8.1 Date: 8.2 Purpose 1: 8.3 Purpose 2: 8.4 Samples: 8.5 Procedure:

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.

AHL assay

Date:

2007/09/15

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:

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

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: