Team:Calgary/Lab/Response

From 2009.igem.org

(Difference between revisions)
Line 83: Line 83:
<br>
<br>
The response circuit consists of the qrr4 promoter followed by the c1 lambda inverter (BBa_Q04510) and <i>aiiA</i> (BBa_C0160). This has been designed and constructed so that in the absence of AI-2, the signalling system acts as a phosphorylation cascade that eventually leads to the phosphorylated form of LuxO, which then complexes with a transcription factor. This complex binds to the qrr4 promoter, inducing the expression of the c1λ repressor protein, thus preventing the expression of aiiA. When AI-2 is present, it binds to LuxP and switches the system into a dephosphorylation cascade, leading to the unphosphorylated form of LuxO. Consequently, no binding occurs at the qrr4 promoter, preventing the expression of the c1λ repressor protein. This allows for the expression of aiiA, a gene that encodes an AHL-degrading enzyme allowing us to target biolfim maintenance.
The response circuit consists of the qrr4 promoter followed by the c1 lambda inverter (BBa_Q04510) and <i>aiiA</i> (BBa_C0160). This has been designed and constructed so that in the absence of AI-2, the signalling system acts as a phosphorylation cascade that eventually leads to the phosphorylated form of LuxO, which then complexes with a transcription factor. This complex binds to the qrr4 promoter, inducing the expression of the c1λ repressor protein, thus preventing the expression of aiiA. When AI-2 is present, it binds to LuxP and switches the system into a dephosphorylation cascade, leading to the unphosphorylated form of LuxO. Consequently, no binding occurs at the qrr4 promoter, preventing the expression of the c1λ repressor protein. This allows for the expression of aiiA, a gene that encodes an AHL-degrading enzyme allowing us to target biolfim maintenance.
 +
<br>
 +
<br>
 +
The functionality of the respose circuit was tested by measuring the fluorscence of response circuit together with LuxO D47E (K218017) mutant, and this fluorscence was compared to the fluorscence of our positive control (R0040 + I13500).
 +
The following is the protocol of the fluorescence reading.
 +
<br>
 +
<br><b>GFP fluorescent reading protocol</b>
 +
<br>1. Grow overnight cultures of each sample
 +
<br>2. Power on the Bio-tec Synergy HT plate reader, or another plate reader, and KC4 application.
 +
<br>3. On a black 96 well plate, aliquot samples in required wells.
 +
<br>4. Go to wizard, and change the reading parameters to the following settings:
 +
<br>Reader: absorbance
 +
<br>Reading type: Endpoint
 +
<br>Wavelength: 570nm (it is as close as it gets to OD600)
 +
<br>5. Click ok.
 +
<br>6. Again, go to wizard, then in layout, mark the wells that contain samples and blank. Click ok.
 +
<br>7. Press the read button
 +
<br>8. Match the OD600 levels by diluting with corresponding Luria-Bertani (LB) broth.
 +
<br>9. Measure OD600 again.
 +
<br>10. Once OD600 are matching for all samples, serial dilute them (1 in 10, 1 in 100). To serial dilute, aliquot 100uL of original culture into a new tube containing 900uL of corresponding LB broth (1 in 10). To make 1 in 100, aliquot 100uL of 1 in 10 dilution into a new tube containing 900uL of corresponding LB broth (1 in 100).
 +
<br>11. Go back to wizard, change the reading parameters to the following settings*:
 +
<br>Reader: Fluorescence
 +
<br>Reading type: Endpoint
 +
<br>Excitation: 485/20
 +
<br>Emission: 528/20
 +
<br>Optics position: Top
 +
<br>Sensitivity: automatic adjustment, scale to high or low well.
 +
<br>Top probe vertical offset: 3mm
 +
<br>12. Click ok.
 +
<br>13. Again, go to wizard, change the layout of the cells.
 +
<br>14. Read.
 +
<br>*GFP reading protocol was obtained from Minenesota State University<br> http://www.mnstate.edu/provost/GFPPlateReaderAssayProtocol.pdf
 +
<br>
<br>
<br>
<br>

Revision as of 06:51, 21 October 2009

University of Calgary

UNIVERSITY OF CALGARY



LAB INDEX
Overview
Updates

Circuits
Quorum Sensing
Chassis Selection

Protocol & Safety
Notebook & Parts
RESPONSE CIRCUIT
The purpose of the response circuit is to show that the AI-2 signalling system can be coupled with a desired response.



Response circuit.png Figure 1. Schematic diagram of the response circuit.

The response circuit consists of the qrr4 promoter followed by the c1 lambda inverter (BBa_Q04510) and aiiA (BBa_C0160). This has been designed and constructed so that in the absence of AI-2, the signalling system acts as a phosphorylation cascade that eventually leads to the phosphorylated form of LuxO, which then complexes with a transcription factor. This complex binds to the qrr4 promoter, inducing the expression of the c1λ repressor protein, thus preventing the expression of aiiA. When AI-2 is present, it binds to LuxP and switches the system into a dephosphorylation cascade, leading to the unphosphorylated form of LuxO. Consequently, no binding occurs at the qrr4 promoter, preventing the expression of the c1λ repressor protein. This allows for the expression of aiiA, a gene that encodes an AHL-degrading enzyme allowing us to target biolfim maintenance.

The functionality of the respose circuit was tested by measuring the fluorscence of response circuit together with LuxO D47E (K218017) mutant, and this fluorscence was compared to the fluorscence of our positive control (R0040 + I13500). The following is the protocol of the fluorescence reading.

GFP fluorescent reading protocol
1. Grow overnight cultures of each sample
2. Power on the Bio-tec Synergy HT plate reader, or another plate reader, and KC4 application.
3. On a black 96 well plate, aliquot samples in required wells.
4. Go to wizard, and change the reading parameters to the following settings:
Reader: absorbance
Reading type: Endpoint
Wavelength: 570nm (it is as close as it gets to OD600)
5. Click ok.
6. Again, go to wizard, then in layout, mark the wells that contain samples and blank. Click ok.
7. Press the read button
8. Match the OD600 levels by diluting with corresponding Luria-Bertani (LB) broth.
9. Measure OD600 again.
10. Once OD600 are matching for all samples, serial dilute them (1 in 10, 1 in 100). To serial dilute, aliquot 100uL of original culture into a new tube containing 900uL of corresponding LB broth (1 in 10). To make 1 in 100, aliquot 100uL of 1 in 10 dilution into a new tube containing 900uL of corresponding LB broth (1 in 100).
11. Go back to wizard, change the reading parameters to the following settings*:
Reader: Fluorescence
Reading type: Endpoint
Excitation: 485/20
Emission: 528/20
Optics position: Top
Sensitivity: automatic adjustment, scale to high or low well.
Top probe vertical offset: 3mm
12. Click ok.
13. Again, go to wizard, change the layout of the cells.
14. Read.
*GFP reading protocol was obtained from Minenesota State University
http://www.mnstate.edu/provost/GFPPlateReaderAssayProtocol.pdf

For an in depth look into the construction of the response circuit (complete with results to date) please read the paper below (or click HERE).
Response Circuit

Retrieved from "http://2009.igem.org/Team:Calgary/Lab/Response"