Template:BOKU-Vienna/Basic Part

Testing of GFP-Ub

In a first round of testing we transformed our ubiquitin tagged GFP into Pichia pastoris under the control of the GAP promoter and the rps25 terminator as shown in the vector map (BB3_A_GAP_GFP_rps25_BC_C). The goal of this experiment was to prove that the gene product is expressed and fluoresces. This picture shows two YPD-Agar plates, the left one overgrown with wild type Pichia pastoris, the right one with the transformed strain, illuminated by UV-light. As you can see the right one glows much brighter in the spots where colonies are growing than the left one.

       <a href="gfp1.png" data-toggle="lightbox">
           <img src="T--BOKU-Vienna--2018_gfp1.png" alt="GFP">
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<a href="gfp2.jpg" data-toggle="lightbox"> 

           <img src="T--BOKU-Vienna--2018_gfp2.jpg" class="w-50" alt="GFP">
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After this first test we used this part in our other constructs as a reporter.

In one instance we used GFP-Ub as a reporter when testing our ethanol construct, represented by the vector map (BB3_A_AlcR_AlcA-GFP_C). In this construct GFP-Ub is expressed in response to the addition of ethanol. This test showed that as the ethanol in the medium is metabolised and evaporates and expression of GFP-Ub halts, the fluorescence goes down again quickly.

<a href="gfp3.png" data-toggle="lightbox"> 

           <img src="T--BOKU-Vienna--2018_gfp3.png" alt="GFP">
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       <a href="gfp4.png" data-toggle="lightbox">
           <img src="T--BOKU-Vienna--2018_gfp4.png" alt="GFP">
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For another experiment that should prove our working toggle switch we used GFP-Ub as a reporter protein again. In these tests it was controlled by a regulatory region featuring a GAP constitutive promoter with four gRNA targets (gTOFF) cloned into it and it was terminated with rps25aTT (BB3_A_gTOFF_GFP_rps25aTT_C). This graph shows the proportion of P. pastoris cells that displayed green fluorescence after 24 (left) and 48 hours (right) of incubation at room temperature. Bars represent the wilson score with the wilson score interval (red) at a error-probability of 5 %. The cells were excited with 488 nm light under a fluorescence microscope and randomly selected sections of them were counted. As you can see in this diagram after 48 h almost 75 % of the observed cells expressed GFP-Ub. Also see the micrograph for an actual impression of these yeast cells.

<a href="gfp5.png" data-toggle="lightbox"> 

           <img src="T--BOKU-Vienna--2018_gfp5.png" alt="GFP">
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       <a href="gfp6.png" data-toggle="lightbox">
           <img src="T--BOKU-Vienna--2018_gfp6.png" alt="GFP">
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           <a href="gfp7.jpg" data-toggle="lightbox">
               <img src="T--BOKU-Vienna--2018_gfp7.jpg" alt="GFP">
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