Part:BBa_K092600:Experience

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Characterization of BBa_K092600

Theoretically this construct should not be able to express RFP due to the fact that the promoter for the induction molecule (TetR) is not present and thus no TetR is expressed.

In the contrary, we found the expression of TetR to be leaky and we carried out characterization of the part. (see protocol in parts registry BBa_K092600).

We found that RPF was indeed produced thus indicating a basal ‘leaky’ expression, and this allows us to predict that upon addition of the PLac promotor, the production of RFP will be highly enhanced.

We obtained a transfer function that is shown below.

Flow cytometry was carried out to verify the presence of RFP expressing cells. After the plate reading assay, the same samples were taken for flow cytometry. Below the flow cytometry for samples at two high concentrations of is shown. In the histograms below, we can clearly see the shift in the different cell types, between those that produce and do not produce RFP. In the x-axis is the log of fluorescence intensity, and in the y-axis the cell count.

These results demonstrate that the construct that was made does indeed function and that upon completion can give high levels of RFP. At the same time such production of RFP can interfere with the precise control that is required in our system.

Protocol

• Three 5 ml cultures of LB medium and antibiotic (ampicillin, 20 µg/ml) were inoculated with single colonies from a glycerol stock stored at –80°C.
• Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm.
• Cultures were diluted 1:1000 into 5 ml of fresh medium and grown to an OD600 of 0.2 under the same conditions as before.
• Twenty-four 100 µl aliquots of each of the cultures were transferred into a flat-bottomed 96 well plate (BD-96).
• Tetracycline stock solution was prepared at a concentration of 6 mg/ml.
• Varying quantities of tetracycline was added to each well to yield 14 different final concentrations. Three replicate wells were measured for each concentration of tetracycline. Three wells were each filled with 200 µl of culture medium to measure the background.
• Plate was left in incubator for 4 hours at 37°C at 60rpm.
• The plate was incubated in a multi-well fluorimeter (Perkin Elmer) at 37°C and assayed with a protocol of fluorescene measurements at 530/580 excitation/emission wavelength filters.
• The transfer function fluorescence intensity after four hours of expression. Error bars representing the 95% confidence interval in the population for the independent samples.
• Flow cytometry was carried out with samples to check for presence of RFP producing cells.

User Reviews

UNIQ669962072cfd5047-partinfo-00000000-QINU

• NTNU_Trondheim 2011

At the beginning of the project our idea was to use this TetR regulated system to build a stress sensor in combination with the rrnB P1 promoter BBa_K639002. However our experience with the biobrick is bad. Transformants did not show any signs of fluorescent activity, and the fragment lengths after enzyme digestion were not as expected.

;

UNIQ669962072cfd5047-partinfo-00000002-QINU