Difference between revisions of "Part:BBa K1893005"

Latest revision as of 21:40, 28 October 2016

Lux receiver with GFP (LuxR+pLux+GFP)

This composite part is based on the Lux quorum-sensing system from Vibrio fischerii. It consists of a Lux receiver device and a GFP reporter that is activated in the presence of a 3O-C6 AHL signal.

Usage and Biology

Quorum sensing is a naturally occurring mechanism that certain strains of bacteria use to regulate gene expression in response to their population density. These bacteria secrete autoinducer signalling molecules, such as N-acyl homoserine lactones (AHLs), that bind to transcription factors to alter gene expression.

In this case, the constitutively expressed LuxR transcriptional regulator (C0062) is activated by the binding of 3O-C6 AHL, an AHL quorum signal. The activated LuxR regulator binds to a LuxR-inducible promoter, pLux, upstream of a GFP reporter. As a result, GFP is expressed when the receiver device is induced with 3O-C6 AHL. We designed this part to characterize the activation range of the Lux receiver device. We are also currently characterising the cross-talk of the Lux receiver device with other quorum-sensing systems, in order to determine its orthogonality.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 985
Illegal BsaI.rc site found at 1712

@@ Line 8: / Line 8: @@
 Quorum sensing is a naturally occurring mechanism that certain strains of bacteria use to regulate gene expression in response to their population density. These bacteria secrete autoinducer signalling molecules, such as N-acyl homoserine lactones (AHLs), that bind to transcription factors to alter gene expression.
-In this case, the constitutively expressed LuxR transcriptional regulator (C0062)  is activated by the binding of 3O-C6 AHL, an AHL quorum signal. The activated LuxR regulator binds to a LuxR-inducible promoter, pLux, upstream of a GFP reporter. As a result, GFP is expressed when the receiver device is induced with 3O-C6 AHL. We designed this part to characterize the activation range of the Lux receiver device. We also characterised the cross-talk of the Lux receiver device with other quorum-sensing systems, in order to determine its orthogonality.
+In this case, the constitutively expressed LuxR transcriptional regulator (C0062)  is activated by the binding of 3O-C6 AHL, an AHL quorum signal. The activated LuxR regulator binds to a LuxR-inducible promoter, pLux, upstream of a GFP reporter. As a result, GFP is expressed when the receiver device is induced with 3O-C6 AHL. We designed this part to characterize the activation range of the Lux receiver device. We are also currently characterising the cross-talk of the Lux receiver device with other quorum-sensing systems, in order to determine its orthogonality.
-===Characterisation===
-The activation range of LuxR by its cognate inducer (3O-C6 AHL) was characterised in TOP10 E.Coli cells. Cells transformed with the Lux response device were cultured to the exponential phase and treated with appropriate concentrations of 3O-C6 AHL in 96-well microplates. These induced cells were grown in the microplates and their fluorescence and absorbance values (OD 600) were monitored over time using a microplate reader.  The reported values for the normalised fluorescence represents the values recorded 180 minutes after AHL induction. The normalised fluorescence was calculated by dividing fluorescence values by absorbance values and correcting for LB autofluorescence.
-In order to characterise the orthogonality of the Lux system, we measured absorbance and fluorescence of the cells in the plate reader after treating them with varying concentrations of 3 different AHL signals (C4 AHL of the Rhl system, 3O-C12 AHL of the Las system and 3O-C14 AHL of the Cin system). This allowed us to determine whether these non-cognate AHLs were capable of activating the LuxR response protein, and therefore the level of crosstalk between the quorum sensing systems.
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