Team:Calgary/Lab/Mutant

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

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Figure 1. Schematic diagram of LuxO D47A (left) and LuxO D47E (right) mutant circuits.

The circuits are almost identical in that they have the same promoter (BBa_R0040), RBS (BBa_B034) and terminator (BBa_B0015). This design will allow for constitutive expression of these proteins. The LuxO D47A mutant mimics the unphosphorylated and thus inactive form of LuxO, meaning that it will not bind to the qrr4 promoter. The LuxO D47E mutant, however, mimics the phosphorylated and thus active form of LuxO, and will thus bind to the qrr4 promoter and induce expression of downstream genes. Once these circuits are tested with a non-Biobrick reporter (in the KT1144 cells) they are used to test whether the reporter circuit is functional. If in the presence of the LuxO D47A mutant the reporter cells do not glow and if in the presence of the LuxO D47E mutant the reporter cells do glow, we know the reporter circuit is functional.

Purpose: To test the functionality of the Reporter and Response circuits. LuxO D47A mimics the unphosphorylated and thus inactive form of LuxO, whereas LuxO D47E mimics the phosphorylated and thus active form of LuxO.

The functionality of the reporter and response circuits were tested by measuring the fluorescence of reporter and response circuits together with LuxO D47E (K218017) mutant, and this fluorescence was compared to the fluorescence 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 Minnesota State University

A paper regarding the construction and testing of the mutant circuits can be read below or HERE.
LuxO Mutants