Tokyo/Works/Assay0

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

Assay0

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-)

*Promoters are inserted into the BioBrick part A-4 with its promoter pluxR deleted (A-4').
[Lux lac hybrid promoter – GFP] is A-4' + lux lac hybrid promoter, for example.

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
The concentrations of AHL and IPTG, if added, were 10 nM and 1 mM, respectively.

Results and Conclusion

Fig.1: Result. Activities of the newly devised lux lac hybrid promoter (hy) were determined in the presence and absence of LacI (lacI(+) and lacI(-), respectively) and those of AHL (AHL(+) and AHL(-)).

Fig.2: Result summary

The newly devised hybrid promoter is activated by AHL and repressed by LacI.

The result is shown in Fig.1 and the characteristics of our hybrid promoter is summarized in Fig. 2.

(1) In the absence of AHL,
fluorescence was as low as that of the negative control, indicating that AHL is necessary to activate this hybrid promoter.
(2) In the presence of AHL,
Fluorescence was the strongest in the absence of LacI, though slightly decreased by IPTG addition.
In the presence of LacI, IPTG addition increased fluorescence up to a quarter of that in the absence of LacI; while, its fluorescence remained the same as that of the negative control in the absence of IPTG. Since it is known that IPTG inhibits repression of LacI, this result indicates that it is LacI that actually represses the hybrid promoter.

Those results indicate that our newly devised hybrid promoter needs AHL for its activation and is repressed by LacI regardless of the presence or absence of AHL. The hybrid promoter is released from LacI repression by IPTG, and activated if there are enough AHL.
Thus, this hybrid promoter is activated
-strongly in the absence of LacI and in the presence of AHL
-moderately in the presence of LacI and IPTG, and in the presence of AHL.