Difference between revisions of "Part:BBa K218011"
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==Characterization of the reporter (Pqrr4 + I13500) circuit==
 
==Characterization of the reporter (Pqrr4 + I13500) circuit==
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The functionality of the reporter was tested by measuring the fluorscence of reporter together with LuxO D47E (K218017) mutant, and this fluorscence was compared to the fluorscence of our positive control (R0040 + I13500).  
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The functionality of the reporter circuit was tested by measuring the fluorscence of reporter 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.
 
The following is the protocol of the fluorescence reading.
 
<br><b>GFP fluorescent reading protocol</b>
 
<br><b>GFP fluorescent reading protocol</b>

Revision as of 06:55, 21 October 2009

LuxO inducible GFP

LuxO when phosphorylated binds to Pqrr4, GFP is expressed. Alternatively, LuxO D47E mutant can also bind to the Pqrr4 and induce GFP production.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 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 945



Characterization of the reporter (Pqrr4 + I13500) circuit

The functionality of the reporter circuit was tested by measuring the fluorscence of reporter 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)