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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.
