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Team:UANL Mty-Mexico/Wetlab
From 2013.igem.org
Wetlab
We divided our circuit in sub-circuits, or modules. Each module comprises a single function of our system. These modules are:
- The mCherry switch.- this switch comprises the 37°C thermometer and an mCherry reporter right downstream from it.
- The GFP switch.- this switch is similar to the mCherry one, but has a 32°C thermometer and a GFP reporter.
- The LacI-GFP switch.- in this switch, a 37°C thermometer is regulating the expression of a LacI gene, which in turn is regulating the expression of a GFP reporter. This GFP reporter is also under the regulation of a 32°C thermometer. In this way, we expect to see two different states: OFF at temperatures below 32°C; ON when temperature is between 32°C and 37°C; and OFF again when temperature is above 37°C.
- The TetR-mCherry switch.- here, an mCherry reporter is regulated by a 37°C thermometer and a pTet promoter; this switch also includes a TetR construction.
- The cI-TetR-mCherry switch.- this switch is similar to the previous one, but also includes a cassette that expresses a thermolabile version of cI. In this way, the expression of mCherry will be ON only at temperatures between 37°C and 42°C and OFF at other temperatures.
Fluorescence Assay
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.
Figure 1. Fluorescence assay for mCherry synthetic construction. The image represent the protocol that we follow to measure the relative fluorescence. We growth the different clone in 25, 30, 37 and 42oC and measure the Fluorescence and OD to normalise the results.
Fluorescence Assay Results
MCherry expression assays regulated with the 37°C thermometer.
From 20 different clones derived from the transformation of the synthetic part in DH5a, four clones were selected based on their differences in color intensity of each colony.
The clones were M1 and M2 with the best expression and M11 and M12 with less or no expression of mCherry protein (M12). Were performed at least three independent experiments on a small scale in microcentrifuge tubes by incubating the samples at 25, 30, 37 and 42°C for 17 hours. Fluorescence was measured for each sample and normalized using the OD of each bacterial culture.
Assay at 25°C.
At that temperature theoretically maintaining the secondary structure of the thermometer. This is reflected in the expression of the clones M1, M2 and M11 (Fig. 2). The three clones have residual expression compared to the control, which is a construction that does not present the thermometer RNA
Test at 30°C.
Similarly, 30°C theoretically thermometer structure remains stable. In fluorescence measurements is recorded a slight increase in the expression of the three clones (Fig. 3).
Test at 37°C.
Instead, rising to 37°C expression increases in all three clones but with different intensity. Theoretically, at this temperature the structure of the thermometer is open. The results shows that the cloned M1 differs from the rest with increased nearly 10-fold relative to the expression at 25°C. While for the M2 is 7X and M11 of only 2X, (Figure 4)
Test at 42°C .
Interestingly, increasing the culture temperature to 42°C , the behavior is practically equal to that recorded at 37oC , which shows us that by opening the structure of the thermometer is kept mCherry expression in both temperatures (Figure 5)
The M12 clone.
One of the clones was selected for having very low expression of mCherry apparent. 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
One of the clones was selected for having very low expression of mCherry apparent. 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
