Difference between revisions of "Part:BBa K218011"
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<br>3. Measure OD600 by using the following settings:
 
<br>3. Measure OD600 by using the following settings:
 
<br>Reader: absorbance
 
<br>Reader: absorbance
−
<br>Still editing.
+
<br>Reading type: Endpoint
 +
<br>Wavelength: 570nm (it is as close as it gets to OD600)
 +
<br>4. From layout, mark the wells that contain samples and blank
 +
<br>5. Press the read button
 +
<br>6. Match the OD600 levels by dilution
 +
<br>7. Once OD600 are matching for all samples, serial dilute them. (1 in 10, 1 in 100)
 +
<br>8. Measure GFP with 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>9. Change the layout of the cells once more.
 +
<br>10. Read
 +
<br>*GFP reading protocol was obtained from Minenesota State University (http://www.mnstate.edu/provost/GFPPlateReaderAssayProtocol.pdf)

Revision as of 04:33, 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


Pictures

Characterization of the reporter (Pqrr4 + I13500) circuit

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). The following is the protocol of the fluorescence reading.
1. Power on the Bio-tec Synergy HT plate reader, or another plate reader, and KC4 application.
2. On a black 96 well plate, aliquot samples in required wells.
3. Measure OD600 by using the following settings:
Reader: absorbance
Reading type: Endpoint
Wavelength: 570nm (it is as close as it gets to OD600)
4. From layout, mark the wells that contain samples and blank
5. Press the read button
6. Match the OD600 levels by dilution
7. Once OD600 are matching for all samples, serial dilute them. (1 in 10, 1 in 100)
8. Measure GFP with 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
9. Change the layout of the cells once more.
10. Read
*GFP reading protocol was obtained from Minenesota State University (http://www.mnstate.edu/provost/GFPPlateReaderAssayProtocol.pdf)