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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.
We present a model for the relation between time, temperature and the change in fluorescence (measured in Relative Fluorescent Units or RFUs) of an E. coli culture that harbors a genetic construction where a fluorescent protein is under control of a RNAT.
Giving the organization of our circuit we will expect the temperature to be the main factor involved in regulation of the fluorescence, where first we will have an off state, followed by an optimal fluorescence and then ending in an off state again, the time will have almost the same effect as it does in any other giving biological phenomena involving gene expression.
Using this model we intend to have the same conditions at the time of the fluorescence measurements so we expect to not have conditions such regarding the incubation (Expect for the temperature) to have a significant effect in the fluorescence.
Although the same conditions will be used the strain used to test the circuit could have and influential effect giving the metabolic and genetic conditions of living.
