Team:UANL Mty-Mexico/Wetlab

From 2013.igem.org

(Difference between revisions)
Line 4: Line 4:
   <li class="active"><a href="#Introduction">Introduction</a></li>
   <li class="active"><a href="#Introduction">Introduction</a></li>
   <li><a href="#mCherry">mCherry thermometer</a></li>
   <li><a href="#mCherry">mCherry thermometer</a></li>
-
   <li><a href="#GFP">GFP thermometer</a></li>
+
   <li><a href="#Conclusions">Conclusions</a></li>
-
  <li><a href="#Conclusions">Transcription factor thermometers</a></li>
+
</ul>
</ul>
Line 46: Line 45:
<p>8. The data was processed with Excel.</p>
<p>8. The data was processed with Excel.</p>
-
 
-
<div id="title">
 
-
<p><a name="GFP"><h3>GFP  <a href="#" class="btn btn-info"><font color="#fff">Back to top</font></a></h3><hr></p></div>
 
<div id="title">
<div id="title">

Revision as of 01:43, 28 September 2013

Carousel Template for Bootstrap


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.

Creative Commons License