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Part:BBa_K1758333

Designed by: Team Bielefeld-CeBiTec 2015   Group: iGEM15_Bielefeld-CeBiTec   (2015-08-30)
Revision as of 03:24, 19 September 2015 by Gila (Talk | contribs)

const. prom+PbrR+PbrA-UTR-sfGFP Lead repressor under control of constitutive promoter and strong RBS Lead repressor under the control of a constitutive promoter with lead induceble promoter and 5´untranslated region in front of a sfGFP for detection.


Usage and Biology

For our biosensor we use parts of the chromosomal lead operon of Cupriavidus metallidurans (Ralstonia metallidurans). The promoter that we use is PbrA. This part of the operon is regulated by the repressor pbrR. The PbrR protein mediates Pb2+-inducible transcription. PbrR belongs to the MerR family, which are metal-sensing regulatory proteins (Borremans et al., 2001). Our sensor system is comprised of PbrR, which is under the control of a constitutive promoter and PbrA as well as a 5’ untranslated region, which controls the transcription of a sfGFP. The sfGFP protein is the measuring output signal.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 465
    Illegal NheI site found at 488
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 640


<h2>To summarize<the in vivo measurments</h2> <p>Our lead sensor was characterized <i>in vivo</i> only. The differences between inductions with various lead concentrations are really slight therefore this sensor needs further optimization which was not possible in this limited time. But as there is a fluorescence response to lead this sensor has the potential work as expected. In the future a characterization in CFPS systems would be interesting.</p> </div>


in vivo characteriation

<p>The <i>pbrAP </i>promoter, the operator box and the PbrR repressor are parts of the chromosomal lead operon of Cupriavidus metallidurans (figure 2). This was cloned and transformed into <i>E.coli </i>KRX. This operon includes now the promoter <i>pbrAP </i>(<a href="https://parts.igem.org/Part:BBa_K1758332" target="_blank"> BBa_K1758332 </a>)), which is regulated by the repressor PbrR. The PbrR belongs to the MerR family, of metal-sensing regulatory proteins, and is Pb2+-inducible. Our sensor system comprises <i>pbrR</i> (<a href="https://parts.igem.org/Part:BBa_K1758330" target="_blank"> BBa_K1758330 </a>) BBa_K1758330 ), which is under the control of a constitutive Promoter and <i>pbrAP</i> and a 5’ untranslated region, which controls the transcription of a sfGFP and increases the fluorescence. Fluorescence implemented by sfGFP protein is the measured output signal (figure 3 and figure 4). </p> 

<figure style="width: 600px">

<a href="Bielefeld-CebiTec_in_vivo_Lead.jpeg" data-lightbox="heavymetals" data-title="The concept of our <i>in vivo</i> lead sensor (<a href="https://parts.igem.org/Part:BBa_K1758332" target="_blank"> BBa_K1758332</a>), which consists of the repressor under the control of a constitutive promoter (<a href="https://parts.igem.org/Part:BBa_K1758330" target="_blank"> BBa_K17583230</a>) and the operator and promoter sequence of the lead inducible promoter. An untranslated region in front of the sfGFP, which is used for detection, enhances its expression (<a href="https://parts.igem.org/Part:BBa_K1758332" target="_blank"> BBa_K1758332</a>)."><img src="Bielefeld-CebiTec_in_vivo_Lead.jpeg" alt="genetical approach"></a> <figcaption>Figure 2: Figure 2: The concept of our in vivo lead sensor (<a href="https://parts.igem.org/Part:BBa_K1758332" target="_blank"> BBa_K1758332</a>)which consists of the repressor under the control of a constitutive promoter (<a href="https://parts.igem.org/Part:BBa_K1758330" target="_blank"> BBa_K17583230</a>) and the operator and promoter sequence of the lead inducible promoter. An untranslated region in front of the sfGFP, which is used for detection, enhances its expression (<a href="https://parts.igem.org/Part:BBa_K1758332" target="_blank"> BBa_K1758332</a>).</figcaption> </figure> 

   <div class="row">

<div class="col-md-6 text-center" style="margin-bottom: 50px"> <figure style="width: 600px"> <a href="http://Bielefeld-CeBiTec_Biolector_lead.jpg" data-lightbox="heavymetals" data-title="Figure 3: Time course of the induction of a lead biosensor with sfGFP for different lead concentrations <i>in vivo</i>. The data are measured with BioLector and normalized to the OD<sub>600</sub>. Error bars represent the standard deviation of two biological replicates. "><img src="Bielefeld-CeBiTec_Biolector_lead.jpg" alt="Adjusting the detection limit"></a> <figcaption>Figure 3: Time course of the induction of a lead biosensor with sfGFP for different lead concentrations <i>in vivo</i>. The data are measured with BioLector and normalized to the OD<sub>600</sub>. Error bars represent the standard deviation of two biological replicates. </figcaption> </figure> </div> <div class="col-md-6 text-center" style="margin-bottom: 50px"><figure style="width: 600px"> <a href="Bielefeld-CeBiTec_Biolector_lead_Balkendiagramm.jpeg" data-lightbox="heavymetals" data-title="Figure 4: Fluorescence levels at three different stages of cultivation. Shown are levels after 60 minutes, 150 minutes and 650 minutes. Error bars represent the standard deviation of two biological replicates. "><img src="Bielefeld-CeBiTec_Biolector_lead_Balkendiagramm.jpeg" alt="Adjusting the detection limit"></a> <figcaption>Figure 4: Fluorescence levels at three different stages of cultivation. Shown are levels after 60 minutes, 150 minutes and 650 minutes. Error bars represent the standard deviation of two biological replicates. </figcaption> </figure> </div> </div>

<p>We tested our lead sensor with sfGFP as reporter gene to verify the functionality of the system. Subsequently, we tested different lead concentrations. The kinetic of our sensors response to different lead concentrations is shown in figure 3. The first 40 hours show a strong increase in fluorescence. After that the increase in fluorescence reaches a plateau. For better visualization the kinetics of figure 3 are represented as bars in figure 4. A fluorescence level difference for 60 min, 150 min and 650 min is represented.</p>


<p> The results of the lead sensor show no significant differences between the different concentrations (figure 3). This might be due to the <i>pbrAP’s</i> weak promoter strength in <i>E. coli.</i> Further reasons are most likely in the weak repressor binding to its operator. So, we suggest for the usage of this sensor, it has to be optimized. Moreover we were lacking time for further in vivo characterizations and different experimental setups. Hence, we did not use this sensor in further experiments regarding Cell-free-Protein-synthesis (CFPS). . In the future a characterization in the CFPS systems would be desirable. </p>

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Categories
//function/sensor/metal
Parameters
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