Team:UANL Mty-Mexico/Wetlab

The part BBa_K110006 which has mCherry under the regulation of the TetR and the RNA Thermometer specific for the 37°C. To verify the operation, a series of experiments were developed in which the bacteria with this part were exposed to different temperatures under the following protocol.

1.- The synthetic constructions were transformed in DH5-alpha and planted in Petri dishes with LB Agar and the corresponding antibiotic.

2.- Twenty different clones were chosen and planted in test tubes with 3 mL and LB medium. They were incubated at 37°C to overnight until saturation.

3.- A visual section of the clones with more and less expression of the Red Fluorescent Protein (mCherry ) was made.

4. 20 μL of the cultivation of all the night were transferred to eppendorf tubes in the microcentrifuge with 500 μL of LB medium and antibiotic. A tiny hole was made to the cap of these tubes with a needle to allow the oxygenation of the cultivation over the experiment.

5.- They were placed at the thermomixer, in eppendorf adjusting the temperature to 25, 37 and 42°C and shaken at seventeen hours at 900 rpm.

6. 200 μl were took from each cultivation and placed in a black 96 well plaque Costar to make measurements in a fluorometer Biotech Synergy HT with the following conditions for mCherry. Excitation filter of 530 +/- 25nm, emission filter of 590 +/- 35 nm, sensibility of 85.

7. The optical density was determined for each cultivation to normalize the measurements reading in a Petri dish with transparent 96 well plaques Costar, with a wave length of 630 nm.

8. The data was processed with Excel.

Fluorescence Assay Results

Of 20 different clones derived from the transformation of E. coli DH5a with the synthetic part BBa_K1140006, four clones were selected based on their differences in colony color intensity.

We called the clones M1, M2, M11 and M12. M1 and M2 showed higher color intensity, while M11 and M12 showed less or no color. At least three independent experiments were performed incubating the samples in microcentrifuge tubes at 25, 30, 37 and 42oC for 17 hours. Fluorescence was measured for each sample and normalized with the OD of each bacterial culture.

At 25ºC, the secondary structure of the thermometer is theoretically maintained. This is reflected in the lower fluorescence of the clones M1, M2 and M11 (Fig. 2). However, the three clones have a residual expression compared to a control without mCherry.

Similarly, at 30ºC the thermometer's structure should remain stable. However, the three clones showed a slight increase in red fluorescence (Fig. 3).

Rising the temperature to 37ºC increases mCherry's expression in all three clones with different intensity. Theoretically, at this temperature the structure of the thermometer is completely open. Nonetheless, expression varies significantly among clones. M1 shows nearly a 10-fold increase in expression relative to that of 25oC. On the other hand, M2 is increased 7X while M11 is increased only 2X (Figure 4).

Increasing the culture temperature to 42ºC does not have any significant effect, which agrees with the assumption that the thermometer retains an open structure (Figure 5) .

One of the clones was selected for apparently having very low expression of mCherry, although it still proved to posses the construction. This was tested at the same time with the clones M1, M2 and M11. Figure 6 shows a comparison between M1 and M12 cloning at all three temperatures. Clearly the clone 12 does not have the behavior of the other three clones.

A comparison between the clones M1, M2 , M11 and M12 at three temperatures can be observed as the clones M1 and M2 have a similar behavior, while M11 has a slight decrease in expression. M12 cloning while not responding to temperature differences. This clone (M12) will be sequenced to find the reason for his behavior. One explanation for this phenomenon is that it possesses a mutation that prevents the expression of mCherry (Figure 7).