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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 RFP switch.- this switch comprises the 37°C thermometer and an RFP reporter right downstream from it.
- The GFP switch.- this switch is similar to the RFP 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-RFP switch.- here, an RFP reporter is regulated by a 37°C thermometer and a pTet promoter; this switch also includes a TetR construction.
- The cI-TetR-RFP 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 RFP will be ON only at temperatures between 37°C and 42°C and OFF at other temperatures.
Fluorescence Essay
The part BBa_K110006 which has RFP under the regulation of the TetR and the RNA Thermometer specific for the 37oC. 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 DH5and 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 37oC to overnight until saturation.
3.- A visual section of the clones with more and less expression of the Red Fluorescent Protein (RFP ) was made.
4. 20 uL of the cultivation of all the night were transferred to eppendorf tubes in the microcentrifuge with 500 uL of LB medium and antibiotic. A tiny hole was made to the cap of these tubes with a needle to allow the aeration of the cultivation over the experiment.
5.- They were placed at the thermomixer, in eppendorf adjusting the temperature to 25, 37 and 42 oC and shaken at seventeen hours at 900rpm.
6. 200 ul were took from each cultivation and placed in boxes of 96 holes and black “Costar” material to make measurements in a fluorometer Biotech Synergy HT with the following conditions for RFP. 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 96 transparent “Costar” holes, with a wave length of 630 nm.
8. The data was processed and graphed using Excel.
