Part:BBa_K218016
LuxO D47E with Constitutive Promoter and RBS upstream
LuxOD47E from Vibrio harveyi with constitutive promoter and RBS.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 216
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 904
Illegal SapI.rc site found at 866
Fluorescent Reading of the Preliminary trial
Figure 1. 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 and a positive control. The left hand 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 followed by an RBS and GFP. TOP10 cells with pBluescript were used as a negative control and to blank the plate reader.
Characterization of the reporter (Pqrr4 + I13500) circuit
The functionality of this reporter circuit was tested by measuring the fluorescence of reporter circuit together with LuxO D47E Mutant Expression Circuit(BBa_K218017), and this fluorescence was compared to the fluorescence of our positive control (BBa_R0040+BBa_I13500). Top 10 E. coli containing the reporter circuit (BBa_K218011) were made chemically competent using standard CaCl2 treatment and then transformed with the LuxOD47E Mutant Expression Circuit (BBa_K218017). Furthermore, in order to test the activity of the LuxO D47E mutant protein (produced from the constructed expression circuit, part BBa_K218017), a standard was obtained from Dr. Bonnie Bassler. Part BBa_K218017 was transformed into the KT1144 E. coli strain containing the Pqrr4-gfp fusion on a cosmid (Bonnie Bassler, Princeton University). Liquid cultures of successful transformants (containing both BBa_K218011 and BBa_K218017) and KT1144 cells transformed with BBa_K21817 were grown overnight (16 hours) along with cultures of BBa_K218011 and BBa_R0040+Bba_I13500 (Positive control; constitutive GFP expression) and pBluescript (Negative control; culture lacking gfp). Overnight cultures as well as 1:10 and 1:100 dilutions and Luria Bertani Media were then aliquoted into a 96 well-plate and readings were taken using the Bio-tec Synergy HT plate reader at 37C. What follows are the detailed instructions for using the Bio-tec Synergy HT 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, date accessed: August 10th, 2009
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