Tokyo/Works/Assay0

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Works top  0. Hybrid promoter  1. Formulation  2. Assay1  3. Simulation  4. Assay2  5. Future works

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:


1-1. pTrc99A + [Lux lac hybrid promoter – GFP] (LacI+) (AHL+)
1-2. pTrc99A + [Lux lac hybrid promoter – GFP] (LacI+) (AHL-)
2-1. pBR322 TetR + [Lux lac hybrid promoter – GFP] (lacI-) (AHL+)
2-2. pBR322 TetR + [Lux lac hybrid promoter – GFP] (lacI-) (AHL-)
3-1. pTrc99A + [placQI – GFP] (placQI is constitutive promoter) (Pos. con.) (AHL+)
3-2. pTrc99A + [placQI – GFP] (placQI is constitutive promoter) (Pos. con.) (AHL-)
4-1. pTrc99A + [LacI promoter – GFP] (Neg. con.) (AHL+)
4-2. pTrc99A + [LacI promoter – GFP] (Neg. con.) (AHL-)
5-1. pBR322 TetR + [LacI promoter – GFP] (Neg. con. check) (AHL+)
5-2. pBR322 TetR + [LacI promoter – GFP] (Neg. con. check) (AHL-)


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

Results and Conclusion

Fig.1: Result
Fig.2: Result summary