Team:Calgary/Lab/Mutant
From 2009.igem.org
(Difference between revisions)
Line 77: | Line 77: | ||
<br> | <br> | ||
</html> | </html> | ||
- | [[Image:mutant_d47a.png|350px]] | + | [[Image:mutant_d47a.png|350px]] [[Image:mutant_d47e.png|350px]] |
- | [[Image:mutant_d47e.png|350px]] | + | |
<html> | <html> | ||
- | <b>Figure 1. Schematic diagram of LuxO D47A (left) and LuxO D47E (right) mutant circuits.</b> 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. | + | <b>Figure 1. Schematic diagram of LuxO D47A (left) and LuxO D47E (right) mutant circuits.</b> |
- | + | <br><br> | |
+ | 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. | ||
+ | <br><br> | ||
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. | 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. | ||
<br> | <br> | ||
<br> | <br> | ||
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 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). | ||
+ | <br> | ||
+ | <br> | ||
The following is the protocol of the fluorescence reading. | The following is the protocol of the fluorescence reading. | ||
<br> | <br> |
Revision as of 07:54, 21 October 2009
UNIVERSITY OF CALGARY