Team:Tsinghua/Project-Summary

From 2013.igem.org

(Difference between revisions)
Line 109: Line 109:
<script type="text/javascript">
<script type="text/javascript">
initialize();
initialize();
-
setupMenu(2, 0);
+
setupMenu(2, 5);
</script>
</script>
</div>
</div>
Line 115: Line 115:
<div id="mycontent">
<div id="mycontent">
<div class="normal">
<div class="normal">
-
<h1>Overview</h1>
+
<h1>Summary</h1>
<p>
<p>
-
            In our project , we take advantage of the mating procedure of yeast to establish a switchable pathogenic microorganism detection box. One of the two haploids—alpha, is designed to play the role as pathogenic sensor, which in function can sense the AHL molecules of pathogenic microorganisms through the novel inducible eukaryotic AHL sensing system designed by our team (PPD sensor).  
+
In our project, we modified pakaryotic quorum sensing system and introduced this system into yeast and thus achieved the sensing of pathogen by yeast. Especially, we reconstructted LuxR by adding three times repeats of VP16 region, as well as a region of nuclear localization sequence (NLS). The repeated sequence of VP16 functions as activation domain (AD) to link the RNA POL II, while the NLS is aimed to import the LuxR to the nuclear and thus enhance the transcriptional activating potency. Applying the mCherry in the downstream of the Lux promoter as the reporter, we verified that our system works with certain efficiency.  
</p>
</p>
<p>
<p>
-
After sense the AHL molecules, the secondary signaling molecules – tetR is produced. The other half, a, is designed to be the reporter which when can output different detectable signals under the stimulation of tetR molecules.
+
We chose Tet-off system as the linker of PPD sensor and PPD reporter, which located in different haploid of yeast. By using the ADE2 knockout strain combined with ADE2 gene at the downstream of Tet operator, we tested that the Tet-off system worked as expected.
</p>
</p>
-
<div class="figure">
 
-
<img class="center" src="https://static.igem.org/mediawiki/2013/e/ec/Tsinghua-Overview1.png" style="width:600px;height:auto;"/>
 
-
</div>
 
<p>
<p>
-
Different types of sensor alpha mate with different types of reporter a, via the Tet-off system, the PPD sensor integrate with PPD reporter and output detectable signal. The work flow within the yeast after mating shows as follows:
+
Finally, we combined the PPD sensor and PPD reporter by mating the haploids.
 +
</p>
 +
<p>
 +
We made the yeast into dry powder and verified that the dry powder made by our protocol can be reactivated after as long as three weeks.
 +
</p>
 +
<h2>Future work</h2>
 +
<p>
 +
So far, we have accomplished all the modules we designed, specifically , we shows that the modified VP16-luxR can bind AHL and recruit POL II and trigger the expression of downstream gene as we detected the expression of mCherry. However, the proportion of yeast with strong flurosence signal reveal to be comparably low. As we use the multiple copy vector of yeast, the pRS423 as vector, we strongly suspected that the difference of plasmid copy number between individual is significant. Thus, the attempt of changing the vector, such as using centromere-like vector will be carried in the future.
 +
</p>
 +
<p>
 +
Secondly, we see few cells in control group with positive mCherry signal. Recall the our plasmid design, as we didn’t add the terminator at the end of each gene, the possibility of leakage between the luxR and the mCherry will in certain degree enhance the background mCherry signal. We plan to add the terminator between luxR and mCherry to optimize our measurement.
 +
</p>
 +
<p>
 +
Thirdly, we proved that the mammalian cell tet-off system we used indeed works in a certain efficiency, that is, when the tetR expressed, it can bind with the TetO and trigger the downstream gene expression. However, as the time limited, we didn’t test if the tetR in different single copy plasmids can also drive the tetO downstream gene expression. We will test this in the future to prove that the mating can be effective and different haploid carried with sensor and reporter can communicate by virtue of tet-off system as expected.
</p>
</p>
-
<div class="figure">
 
-
<img class="center" src="https://static.igem.org/mediawiki/2013/2/26/Tsinghua-Overview2.jpg" style="width:600px;height:auto;"/>
 
-
</div>
 
<p>
<p>
-
After mating, the specific “pathogenic detector” will be transformed into dry powder, which is the nonbioactivity form of yeast and can be stored under normal condition for weeks. The test paper can be produced by stick the yeast dry powder into specific paper with growth medium and activated by water. Special biology sample can be added on to the test paper and by visible signal we can conclude if the people have infected with one type of microorganism.
+
Finally, we have tested if the yeast strain we used can be made into the dry powder and can be reactivated several days later just as baker yeast. Our results successfully show that the method we used to deal with the yeast dry powder indeed retain its activity for at least 3 weeks, yet we need to further prove that the method we raised to produce the test paper can also works. That is, if we put the grow medium in the form of powder can also support the yeast to grow.
</p>
</p>
</div>
</div>

Revision as of 19:29, 27 September 2013

Summary

In our project, we modified pakaryotic quorum sensing system and introduced this system into yeast and thus achieved the sensing of pathogen by yeast. Especially, we reconstructted LuxR by adding three times repeats of VP16 region, as well as a region of nuclear localization sequence (NLS). The repeated sequence of VP16 functions as activation domain (AD) to link the RNA POL II, while the NLS is aimed to import the LuxR to the nuclear and thus enhance the transcriptional activating potency. Applying the mCherry in the downstream of the Lux promoter as the reporter, we verified that our system works with certain efficiency.

We chose Tet-off system as the linker of PPD sensor and PPD reporter, which located in different haploid of yeast. By using the ADE2 knockout strain combined with ADE2 gene at the downstream of Tet operator, we tested that the Tet-off system worked as expected.

Finally, we combined the PPD sensor and PPD reporter by mating the haploids.

We made the yeast into dry powder and verified that the dry powder made by our protocol can be reactivated after as long as three weeks.

Future work

So far, we have accomplished all the modules we designed, specifically , we shows that the modified VP16-luxR can bind AHL and recruit POL II and trigger the expression of downstream gene as we detected the expression of mCherry. However, the proportion of yeast with strong flurosence signal reveal to be comparably low. As we use the multiple copy vector of yeast, the pRS423 as vector, we strongly suspected that the difference of plasmid copy number between individual is significant. Thus, the attempt of changing the vector, such as using centromere-like vector will be carried in the future.

Secondly, we see few cells in control group with positive mCherry signal. Recall the our plasmid design, as we didn’t add the terminator at the end of each gene, the possibility of leakage between the luxR and the mCherry will in certain degree enhance the background mCherry signal. We plan to add the terminator between luxR and mCherry to optimize our measurement.

Thirdly, we proved that the mammalian cell tet-off system we used indeed works in a certain efficiency, that is, when the tetR expressed, it can bind with the TetO and trigger the downstream gene expression. However, as the time limited, we didn’t test if the tetR in different single copy plasmids can also drive the tetO downstream gene expression. We will test this in the future to prove that the mating can be effective and different haploid carried with sensor and reporter can communicate by virtue of tet-off system as expected.

Finally, we have tested if the yeast strain we used can be made into the dry powder and can be reactivated several days later just as baker yeast. Our results successfully show that the method we used to deal with the yeast dry powder indeed retain its activity for at least 3 weeks, yet we need to further prove that the method we raised to produce the test paper can also works. That is, if we put the grow medium in the form of powder can also support the yeast to grow.

Retrieved from "http://2013.igem.org/Team:Tsinghua/Project-Summary"