Team:Tsinghua/Project-Sensor

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<h1>PPD Sensor</h1>
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<h2>Overview</h2>
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<p>
 +
  We constructed the PPD sensor part by reconstructing bacteria quorum sensing system in yeast.
 +
  </p>
 +
<p>
 +
  We confirm that the Lux Receptor in the sensor was expressed.
 +
  </p>
 +
<p>
 +
  We realized the inducing regulation of our sensor part by adding AHL to the yeast.
 +
  </p>
 +
<h2>Designed PPD Sensor Part</h2>
 +
<p>
 +
  To achieve the sensing of the AHLs from the pathogenic organisms, we plan to introduce the qurum sensing system to yeast, which has been naturally used by the pathogenic microoganisms themselves for communicating within the colonies. Usually, the quorum sensing system share the similar working mechanisms between different species, the precise procedures are shown as follows:
 +
  </p>
 +
<div class="figure">
 +
<img class="center" src="https://static.igem.org/mediawiki/2013/f/f7/Tsinghua-sensor1.png"/>
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<p class="legend">
 +
    Figure 1.
 +
    </p>
 +
</div>
 +
<h2>Reconstruction the PPD System</h2>
 +
<p>
 +
  Considered the difference between prokaryotic and eukaryotic system, the prokaryotic quorum sensing system needs to be reconstructed in a “eukaryotic way” when introduced into yeast. As the quorum sensing systems in different species share the similar mechanism, we typically reconstructed the LuxR system and further test the efficiency of the modified system.
 +
  </p>
 +
<div class="figure">
 +
<img class="center" src="https://static.igem.org/mediawiki/2013/f/f6/Tsinghua-sensor2.png"/>
 +
<p class="legend">
 +
    Figure 2.
 +
    </p>
 +
</div>
 +
<p>
 +
  For the reconstruction of LuxR, we add three times repeat of VP16 region, as well as a region of NLS. The 3 times repeated sequence of VP16 is function as activation domain (AD) to link the RNA POL II, the Nuclear Localization Sequence (NLS) is aimed to import the LuxR to the nuclear and thus enhance the transcriptional activating potency.
 +
  </p>
 +
<p>
 +
  For the reconstruction of pLux Promoter, we add cyc100 mini promoter (the TATA Box), the function of which is to recruit RNA POL II to DNA region.
 +
  </p>
 +
<p>
 +
  These modifications will lead to the effect that when AHL is introduced into the cell, LuxR will bind with AHL and then together bind with pLux promoter, the VP16 domain of the complex will then bind with POL II, which bind to cyc100 mini promoter and facilitate the gene expression downstream of plux+cyc100 promoter.
 +
  </p>
 +
<p>
 +
  To test the function of PPD sensor part, we used mCherry to report the transcription ability of cyc100 mini promoter.
 +
  </p>
 +
<div class="figure">
 +
<img class="center" src="https://static.igem.org/mediawiki/2013/0/00/Tsinghua-sensor3.png"/>
 +
<p class="legend">
 +
    Figure 3.
 +
    </p>
 +
</div>
 +
<h2>PPD Sensor System Constructing</h2>
 +
<p>
 +
    We designed a basic part in our sensor system and synthesized it. The map of the part is shown below. The main part contains triple VP16, Nuclear Location Sequence (NLS), LuxR, pLux and cyc100 mini promoter.
 +
  </p>
 +
<div class="figure">
 +
<img class="center" src="https://static.igem.org/mediawiki/2013/2/21/Tsinghua-sensor4.png"/>
 +
<p class="legend">
 +
    Figure 4. Synthesized plasmid containing tVP16-LuxR-Plux-cyc100 mini promoter
 +
    </p>
 +
</div>
 +
<p>To transfer the system into yeast, we cloned the synthesized part into pRS423, a multi-copy expression vector used in yeast. The pMV-LuxR vector and pRS423 was cut by SpeI、BamHI. Result is shown in Figure 5.
 +
  </p>
 +
<div class="figure">
 +
<img class="center" src="https://static.igem.org/mediawiki/2013/5/54/Tsinghua-sensor5.png"/>
 +
<p class="legend">
 +
    Figure 5. Restriction enzyme cutting result for Luxr_ZHU_MENG and pRS423
 +
    </p>
 +
</div>
 +
<p>LuxR_ZHU_Meng fragment was ligased with pRS423 fragment. We got the following Clone1 plasmid.
 +
  </p>
 +
<div class="figure">
 +
<img class="center" src="https://static.igem.org/mediawiki/2013/f/fc/Tsinghua-sensor6.png"/>
 +
<p class="legend">
 +
    Figure 6. Clone1
 +
    </p>
 +
</div>
 +
<p>Then we added TEF promoter before LuxR_ZHU_MENG part. As shown in the following picture, pTEF and Clone1 plasmid was digested by SacI and SpeI. Then the two fragments were ligased to construct Clone2 plasmid.
 +
  </p>
 +
<div class="figure">
 +
<img class="center" src="https://static.igem.org/mediawiki/2013/8/86/Tsinghua-sensor7.png"/>
 +
<p class="legend">
 +
    Figure 7. Restriction enzyme cutting result for pTEF and Clone1
 +
    </p>
 +
</div>
 +
<div class="figure">
 +
<img class="center" src="https://static.igem.org/mediawiki/2013/9/98/Tsinghua-sensor8.png"/>
 +
<p class="legend">
 +
    Figure 8. Clone2
 +
    </p>
 +
</div>
 +
<p>Then, we insert a mCherry after the cyc100 mini promoter as a reporter for the sensor system. We used BamHI and EcoRI to digest the Clone2 and mCherry PCR product. pTF4 was constructed by ligasing these two fragments.
 +
  </p>
 +
<div class="figure">
 +
<img class="center" src="https://static.igem.org/mediawiki/2013/a/a4/Tsinghua-sensor9.png"/>
 +
<p class="legend">
 +
    Figure 9. Restriction enzyme cutting result for Clone2 and mCherry
 +
    </p>
 +
</div>
 +
<div class="figure">
 +
<img class="center" src="https://static.igem.org/mediawiki/2013/a/a6/Tsinghua-sensor10.png"/>
 +
<p class="legend">
 +
    Figure 10. pTF4
 +
    </p>
 +
</div>
 +
<h2>PPD sensor system testing</h2>
 +
<p>We tested the feasibility of our PPD sensor system.</p>
 +
<p>At first, we wanted to detect whether LuxR protein was expressed in the yeast. The tVP16-LucR protein was tagged by flag. We transformed the pTF4 into yeast and cultured for 24h before being lysed by 0.2 M NaOH. The yeast total protein was treated by SDS sample buffer. The samples, as well as the control group, were separated by SDS-PAGE and detected by anti-flag antibody. Result is show below.
 +
  </p>
 +
<div class="figure">
 +
<img class="center" src="https://static.igem.org/mediawiki/2013/4/40/Tsinghua-sensor11.png"/>
 +
<p class="legend">
 +
    Figure 11. Expression of tVP16-LuxR in yeast
 +
    </p>
 +
</div>
 +
<p>From Figure 11, we can know that compared with control group, pTF4 transformed yeast had higher expression level of tVP16-LuxR. However, it seems that there is a week band in control group. We speculated that it might be caused by non-specific binding of anti-flag first antibody.
 +
  </p>
 +
<p>
 +
  Then we examine the transcription potency of the PPD. We transformed the pTF4 into yeast. One group was treated with 0.5 μM AHL, while the other not. After 24 h inducing, compared with control group, the AHL inducing group had more mCherry positive cells. The several mCherry cells in the control group might be caused by the leak of the pLux-cyc100 mini promoter.
 +
  </p>
 +
<div class="figure">
 +
<img class="center" src="https://static.igem.org/mediawiki/2013/b/ba/Tsinghua-sensor12.png"/>
 +
<p class="legend">
 +
    Figure 12. PPD sensor induced by 0.5 uM AHL
 +
    </p>
 +
</div>
 +
<p>The efficiency of expression had been quantified via flow cytometry. This time, we set three groups. One group was treated with 0.5 μM AHL. The second group was treated with 0.5 μM with PEG, which was used to increase the fluidity of yeast membrane. Last group was control. We detected the mCherry signals 0.5h, 2h, 8h, and 24h after AHL treatment. The results are shown below.
 +
  </p>
 +
<div class="figure">
 +
<img class="center" src="https://static.igem.org/mediawiki/2013/0/03/Tsinghua-sensor13.png"/>
 +
<p class="legend">
 +
    Figure 13. Flow cytometry analysis of AHL inducing result in different time points
 +
    </p>
 +
</div>
 +
<p>
 +
  From the result, we can conclude that, after 24h inducing, the mCherry expression rate in AHL group is slight higher than that in control group. mCherry signal in 24h AHL+PEG was nearly negative, because the PEG may do harm to the survival of yeasts. Unfortunately, in earlier time points, we could hardly detect mCherry signal.
 +
  </p>
 +
<p>
 +
  To improve the system, we increased the AHL concentration to 750 μM
 +
  </p>
 +
<h2>Discussion</h2>
 +
</div>
 +
</div>
 +
</div>
 +
</body></html>

Revision as of 04:11, 26 September 2013

PPD Sensor

Overview

We constructed the PPD sensor part by reconstructing bacteria quorum sensing system in yeast.

We confirm that the Lux Receptor in the sensor was expressed.

We realized the inducing regulation of our sensor part by adding AHL to the yeast.

Designed PPD Sensor Part

To achieve the sensing of the AHLs from the pathogenic organisms, we plan to introduce the qurum sensing system to yeast, which has been naturally used by the pathogenic microoganisms themselves for communicating within the colonies. Usually, the quorum sensing system share the similar working mechanisms between different species, the precise procedures are shown as follows:

Figure 1.

Reconstruction the PPD System

Considered the difference between prokaryotic and eukaryotic system, the prokaryotic quorum sensing system needs to be reconstructed in a “eukaryotic way” when introduced into yeast. As the quorum sensing systems in different species share the similar mechanism, we typically reconstructed the LuxR system and further test the efficiency of the modified system.

Figure 2.

For the reconstruction of LuxR, we add three times repeat of VP16 region, as well as a region of NLS. The 3 times repeated sequence of VP16 is function as activation domain (AD) to link the RNA POL II, the Nuclear Localization Sequence (NLS) is aimed to import the LuxR to the nuclear and thus enhance the transcriptional activating potency.

For the reconstruction of pLux Promoter, we add cyc100 mini promoter (the TATA Box), the function of which is to recruit RNA POL II to DNA region.

These modifications will lead to the effect that when AHL is introduced into the cell, LuxR will bind with AHL and then together bind with pLux promoter, the VP16 domain of the complex will then bind with POL II, which bind to cyc100 mini promoter and facilitate the gene expression downstream of plux+cyc100 promoter.

To test the function of PPD sensor part, we used mCherry to report the transcription ability of cyc100 mini promoter.

Figure 3.

PPD Sensor System Constructing

We designed a basic part in our sensor system and synthesized it. The map of the part is shown below. The main part contains triple VP16, Nuclear Location Sequence (NLS), LuxR, pLux and cyc100 mini promoter.

Figure 4. Synthesized plasmid containing tVP16-LuxR-Plux-cyc100 mini promoter

To transfer the system into yeast, we cloned the synthesized part into pRS423, a multi-copy expression vector used in yeast. The pMV-LuxR vector and pRS423 was cut by SpeI、BamHI. Result is shown in Figure 5.

Figure 5. Restriction enzyme cutting result for Luxr_ZHU_MENG and pRS423

LuxR_ZHU_Meng fragment was ligased with pRS423 fragment. We got the following Clone1 plasmid.

Figure 6. Clone1

Then we added TEF promoter before LuxR_ZHU_MENG part. As shown in the following picture, pTEF and Clone1 plasmid was digested by SacI and SpeI. Then the two fragments were ligased to construct Clone2 plasmid.

Figure 7. Restriction enzyme cutting result for pTEF and Clone1

Figure 8. Clone2

Then, we insert a mCherry after the cyc100 mini promoter as a reporter for the sensor system. We used BamHI and EcoRI to digest the Clone2 and mCherry PCR product. pTF4 was constructed by ligasing these two fragments.

Figure 9. Restriction enzyme cutting result for Clone2 and mCherry

Figure 10. pTF4

PPD sensor system testing

We tested the feasibility of our PPD sensor system.

At first, we wanted to detect whether LuxR protein was expressed in the yeast. The tVP16-LucR protein was tagged by flag. We transformed the pTF4 into yeast and cultured for 24h before being lysed by 0.2 M NaOH. The yeast total protein was treated by SDS sample buffer. The samples, as well as the control group, were separated by SDS-PAGE and detected by anti-flag antibody. Result is show below.

Figure 11. Expression of tVP16-LuxR in yeast

From Figure 11, we can know that compared with control group, pTF4 transformed yeast had higher expression level of tVP16-LuxR. However, it seems that there is a week band in control group. We speculated that it might be caused by non-specific binding of anti-flag first antibody.

Then we examine the transcription potency of the PPD. We transformed the pTF4 into yeast. One group was treated with 0.5 μM AHL, while the other not. After 24 h inducing, compared with control group, the AHL inducing group had more mCherry positive cells. The several mCherry cells in the control group might be caused by the leak of the pLux-cyc100 mini promoter.

Figure 12. PPD sensor induced by 0.5 uM AHL

The efficiency of expression had been quantified via flow cytometry. This time, we set three groups. One group was treated with 0.5 μM AHL. The second group was treated with 0.5 μM with PEG, which was used to increase the fluidity of yeast membrane. Last group was control. We detected the mCherry signals 0.5h, 2h, 8h, and 24h after AHL treatment. The results are shown below.

Figure 13. Flow cytometry analysis of AHL inducing result in different time points

From the result, we can conclude that, after 24h inducing, the mCherry expression rate in AHL group is slight higher than that in control group. mCherry signal in 24h AHL+PEG was nearly negative, because the PEG may do harm to the survival of yeasts. Unfortunately, in earlier time points, we could hardly detect mCherry signal.

To improve the system, we increased the AHL concentration to 750 μM

Discussion

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