Tokyo/Works/Assay0

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__NOTOC__
__NOTOC__
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<br>[[Tokyo/Works|Works top]]　　0. [[Tokyo/Works/Hybrid promoter|Hybrid promoter]]　　1. [[Tokyo/Works/Formulation |Formulation]]　　2. [[Tokyo/Works/Assay |Assay1]]　　3. [[Tokyo/Works/Simulation |Simulation]]　　4. [[Tokyo/Works/Assay2 |Assay2]]　　5. [[Tokyo/Works/Future works |Future works]]
+
<br>[[Tokyo/Works|Works top]]　　'''0. [[Tokyo/Works/Hybrid promoter|Hybrid promoter]]'''　　1. [[Tokyo/Works/Formulation |Formulation]]　　2. [[Tokyo/Works/Assay |Assay1]]　　3. [[Tokyo/Works/Simulation |Simulation]]　　4. [[Tokyo/Works/Assay2 |Assay2]]　　5. [[Tokyo/Works/Future works |Future works]]
 +
<br>[[Tokyo/Works/Assay0|Assay0]]
===Purpose 1:===  
===Purpose 1:===  
To check if LacI hybrid promoter is activated by AHL and repressed by LacI.
To check if LacI hybrid promoter is activated by AHL and repressed by LacI.
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===Samples: ===
===Samples: ===
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<br>1-1. pTrc99A + [Lux lac hybrid promoter – GFP] '''(LacI+) (AHL+)'''
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<br>1-1. pTrc99A + [Lux lac hybrid promoter – GFP] ([http://partsregistry.org/Part:BBa_I751900 BBa_I751900]) '''(LacI+) (AHL+)'''
-
<br>1-2. pTrc99A + [Lux lac hybrid promoter – GFP] '''(LacI+) (AHL-)'''
+
<br>1-2. pTrc99A + [Lux lac hybrid promoter – GFP] ([http://partsregistry.org/Part:BBa_I751900 BBa_I751900]) '''(LacI+) (AHL-)'''
-
<br>2-1. pBR322 TetR + [Lux lac hybrid promoter – GFP] '''(lacI-) (AHL+)'''
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<br>2-1. pBR322 TetR + [Lux lac hybrid promoter – GFP] ([http://partsregistry.org/Part:BBa_I751900 BBa_I751900]) '''(lacI-) (AHL+)'''
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<br>2-2. pBR322 TetR + [Lux lac hybrid promoter – GFP] '''(lacI-) (AHL-)'''
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<br>2-2. pBR322 TetR + [Lux lac hybrid promoter – GFP] ([http://partsregistry.org/Part:BBa_I751900 BBa_I751900]) '''(lacI-) (AHL-)'''
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<br>3-1. pTrc99A + [placQI – GFP] (placQI is constitutive promoter) (Pos. con.) (AHL+)
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<br>3-1. pTrc99A + [placIQ(constitutive) – GFP] ([http://partsregistry.org/Part:BBa_J54201 BBa_J54201]) (Pos. con.) (AHL+)
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<br>3-2. pTrc99A + [placQI – GFP] (placQI is constitutive promoter) (Pos. con.) (AHL-)
+
<br>3-2. pTrc99A + [placIQ(constitutive) – GFP] ([http://partsregistry.org/Part:BBa_J54201 BBa_J54201]) (Pos. con.) (AHL-)
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<br>4-1. pTrc99A + [LacI promoter – GFP] (Neg. con.) (AHL+)
+
<br>4-1. pTrc99A + [A4 ΔP] ([http://partsregistry.org/Part:BBa_I751100 BBa_I751100])<!-- [LacI promoter – GFP] --> (Neg. con.) (AHL+)
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<br>4-2. pTrc99A + [LacI promoter – GFP] (Neg. con.) (AHL-)
+
<br>4-2. pTrc99A + [A4 ΔP] ([http://partsregistry.org/Part:BBa_I751100 BBa_I751100])<!--[LacI promoter – GFP] -->(Neg. con.) (AHL-)
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<br>5-1. pBR322 TetR + [LacI promoter – GFP] (Neg. con. check) (AHL+)
+
-
<br>5-2. pBR322 TetR + [LacI promoter – GFP] (Neg. con. check) (AHL-)
+
 +
<!-- <br>5-1. pBR322 TetR + [LacI promoter – GFP]  (Neg. con. check) (AHL+)
 +
<br>5-2. pBR322 TetR + [LacI promoter – GFP]  (Neg. con. check) (AHL-)
 +
-->
 +
 +
<!--
 +
<br>*Promoters are inserted into the BioBrick part A-4 with its promoter pluxR deleted (A-4').
 +
<br>[Lux lac hybrid promoter – GFP] is A-4' + lux lac hybrid promoter, for example.
 +
-->
<br>'''Repressor LacI expression:'''  
<br>'''Repressor LacI expression:'''  
<br>pTrc99A expresses LacI
<br>pTrc99A expresses LacI
-
<br>pBR322 TetR does NOT express LacI
+
<br>pBR322 TetR does NOT express LacI (but expresses TetR, so the condition is more similar than "empty" DH5alpha)
<br>'''Antibiotics resistance:'''  
<br>'''Antibiotics resistance:'''  
<br>pTrc99A gives ampicillin-resistance
<br>pTrc99A gives ampicillin-resistance
<br>pBR322 TetR gives ampicillin-resistance
<br>pBR322 TetR gives ampicillin-resistance
-
<br>[LacI hybrid promoter – GFP] gives kanamicin-resistance
+
<br>[LacI hybrid promoter – GFP] ([http://partsregistry.org/Part:BBa_I751900 BBa_I751900]) gives kanamicin-resistance
-
<br>[placQI – GFP] gives kanamicin-resistance
+
<br>[placIQ – GFP] ([http://partsregistry.org/Part:BBa_J54201 BBa_J54201])  gives kanamicin-resistance
===Procedure:===
===Procedure:===
<br>[[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
<br>[[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
 +
<br>The concentrations of AHL and IPTG, if added, were 10 nM and 1 mM, respectively.
===Results and Conclusion===
===Results and Conclusion===
[[Image:hybridgraph2.JPG|thumb|350px| '''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(-)).]]
[[Image:hybridgraph2.JPG|thumb|350px| '''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(-)).]]
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[[Image:hybridgraph.JPG|thumb|300px| '''Fig.2: '''Result summary]]
+
[[Image:hybridgraph3.JPG|thumb|300px| '''Fig.2: '''Result summary]]
<br>
<br>
'''The newly devised hybrid promoter is activated by AHL and repressed by LacI.'''  
'''The newly devised hybrid promoter is activated by AHL and repressed by LacI.'''  
<br><br>
<br><br>
-
The result is shown in Fig.1 and the characteristics of our hybrid promoter is summarized in Fig. 2.<br>
+
The result is shown in '''Fig.1''' and the characteristics of our hybrid promoter is summarized in '''Fig. 2'''.<br>
<br>(1) In the absence of AHL,  
<br>(1) In the absence of AHL,  
<br>
<br>
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<br>
<br>
Thus, this hybrid promoter is activated  
Thus, this hybrid promoter is activated  
-
-strongly in the absence of LacI and in the presence of AHL
+
<br>-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.
+
<br>-moderately in the presence of LacI and IPTG, and in the presence of AHL.

Latest revision as of 19:57, 26 October 2007


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] ([http://partsregistry.org/Part:BBa_I751900 BBa_I751900]) (LacI+) (AHL+)
1-2. pTrc99A + [Lux lac hybrid promoter – GFP] ([http://partsregistry.org/Part:BBa_I751900 BBa_I751900]) (LacI+) (AHL-)
2-1. pBR322 TetR + [Lux lac hybrid promoter – GFP] ([http://partsregistry.org/Part:BBa_I751900 BBa_I751900]) (lacI-) (AHL+)
2-2. pBR322 TetR + [Lux lac hybrid promoter – GFP] ([http://partsregistry.org/Part:BBa_I751900 BBa_I751900]) (lacI-) (AHL-)
3-1. pTrc99A + [placIQ(constitutive) – GFP] ([http://partsregistry.org/Part:BBa_J54201 BBa_J54201]) (Pos. con.) (AHL+)
3-2. pTrc99A + [placIQ(constitutive) – GFP] ([http://partsregistry.org/Part:BBa_J54201 BBa_J54201]) (Pos. con.) (AHL-)
4-1. pTrc99A + [A4 ΔP] ([http://partsregistry.org/Part:BBa_I751100 BBa_I751100]) (Neg. con.) (AHL+)
4-2. pTrc99A + [A4 ΔP] ([http://partsregistry.org/Part:BBa_I751100 BBa_I751100])(Neg. con.) (AHL-)



Repressor LacI expression:
pTrc99A expresses LacI
pBR322 TetR does NOT express LacI (but expresses TetR, so the condition is more similar than "empty" DH5alpha)


Antibiotics resistance:
pTrc99A gives ampicillin-resistance
pBR322 TetR gives ampicillin-resistance
[LacI hybrid promoter – GFP] ([http://partsregistry.org/Part:BBa_I751900 BBa_I751900]) gives kanamicin-resistance
[placIQ – GFP] ([http://partsregistry.org/Part:BBa_J54201 BBa_J54201]) 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.

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