Team:Calgary/Lab/Reporter

• Signalling Circuit
• Reporter Circuit
• Mutant Circuits
• Response Circuit

The purpose of the reporter circuit is to test the functionality of the signalling circuit.

Figure 1. Schematic diagram of reporter circuit. The reporter circuit is used to test whether the signalling circuit is functional. It has been designed and constructed with the qrr4 promoter followed by GFP. In the absence of AI-2, the end product of the signalling cascade is phospho-LuxO, which will bind to the qrr4 promoter and therefore produce fluorescence. In the presence of AI-2 however, the end product of the signalling cascade is LuxO, which will not bind to the qrr4 promoter, and therefore the colonies will not glow.

CHARACTERIZATION
The functionality of the reporter circuit was tested by transforming it into the same cell as the LuxO D47E (K218017) mutant and then measuring fluorescence. The expectation was such that without LuxO D47E, the reporter cells would have basal levels of fluorescence, but upon the addition of the mutant, fluorescence would increase. This is because LuxO D47E mimics the phosphorylated and thus active form of LuxO and should bind to the qrr4 promoter and induce expression of GFP. Below depicts the results of this fluorescent readings and the protocol can be found under the figure.

Figure 2. Fluorescent readings when testing LuxO D47E mutants in KT1144 cells and testing the reporter circuit with functional LuxO D47E mutants. This graph is divided into two lines of cells with a positive control. The left hadn bars depict the KT1144 cells with and without LuxO D47E, and this test shows that the mutant is functional because there is an increase in fluorescence upon the addition of the mutant. See 'mutant circuits' on the side bar for more information on testing. The second line of cells is the reporter circuit with and without LuxO D47E, and the purpose here is to determine whether the reporter circuit is functional. Without the mutant circuit, fluorescence reads at 6699, whereas with the mutant, fluorescence reads at 12699. As there is an increase in fluorescence upon the addition of the LuxO D47E mutant, the reporter circuit is functional. The positive control is the TetR promoter followd by an RBS and GFP. TOP10 cells with pBluescript were used as a negative control and to blank the plate reader

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 and testing of reporter circuit (complete with results) please read the paper below.