Difference between revisions of "Part:BBa K218011"

Latest revision as of 22:31, 24 October 2009

LuxO inducible GFP

This circuit acts as a reporter for the LuxO protein (see Part:BBa_K218001). When LuxO phosphorylated and thus active, phospho-LuxO complexes with transcrition factor 54 and binds to the qrr4 promoter, inducing expression of GFP. Alternatively, the LuxO D47E mutant (that mimics phospho-LuxO and is thus active, Part:BBa_K218017) can also bind to Pqrr4 and induce GFP expression.

Sequence and Features

Preliminary Fluorescent Readings to Test Circuit

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. The increase in fluorescence is due to the fact that the LuxO D47E mutant mimics the phosphorylated and thus active form of LuxO and will bind to the qrr4 promoter and induce expression of GFP. The second line of cells is the reporter circuit with and without the LuxO D47E circuit, 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 construct was tested by transforming the LuxO D47E mutant(Part:BBa_K218017) into the same cell as the reporter and then measuring fluorescence. In order to verify that this circuit was functional, we first had to ensure that the mutant circuit was functional so it could be used to test the reporter. The mutant circuit was transformed into the chemically competent KT1144 E. coli strain containing the Pqrr4-gfp fusion on a cosmid (Bonnie Bassler, Princeton University). In parallel, TOP 10 E. coli containing the reporter circuit (BBa_K218011) were made chemically competent following standard Calcium Chloride treatment protocols. The LuxOD47E mutant was then transformed into these cells. Liquid cultures of (1) the mutant circuit in the KT1144 cells and (2)the mutant circuit in E. coli with the reporter 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 37 degrees Celsius. Detailed instructions for using the Bio-tec Synergy HT plate reader are listed below.

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