Part:BBa_K1758330

Lead repressor under control of constitutive promoter and strong RBS

Lead promoter repressor under the control of konstitutive promoter (K608002)

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
  COMPATIBLE WITH RFC[1000]

h2><i>in vivo characterization </i></h2></br>

In addition to the other heavy metal sensors, we constructed a sensor for lead detection. It consists of the repressor PbrR which binds at the operator box downstream of the pbrAP promoter. The binding of the repressor is reversible in the presence of Pb²⁺ Ions. Those ions can weakened the repressors binding and hence, all genes downstream of the pbrAP promoter can be expressed. Like the former sensors this one encloses a sfGFP for detection via fluorescence. So if no lead is present in the media, the repressor binds to the operator box and the pbrAP promoter is blocked meaning that the transcription of sfGFP is prevented. No fluorescence signal is the results. By supplementation of lead, the repressor is separated from the operator box and the genes downstream of the promoter can be expressed.

The pbrAP 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 E.coli KRX. This operon includes now the promoter pbrAP (<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 pbrR (<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 pbrAP 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).

<figure style="width: 600px">

<a href="" data-lightbox="heavymetals" data-title="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>)."><img src="" 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>

<figure style="width: 600px">

<a href="http://" data-lightbox="heavymetals" data-title="Figure 3: Time course of the induction of a lead biosensor with sfGFP for different lead concentrations in vivo. The data are measured with BioLector and normalized to the OD₆₀₀. Error bars represent the standard deviation of two biological replicates. "><img src="" alt="Adjusting the detection limit"></a> <figcaption>Figure 3: Time course of the induction of a lead biosensor with sfGFP for different lead concentrations in vivo. The data are measured with BioLector and normalized to the OD₆₀₀. Error bars represent the standard deviation of two biological replicates. </figcaption> </figure>

<figure style="width: 600px">

<a href="" 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="" 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>

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.

The results of the lead sensor show no significant differences between the different concentrations (figure 3). This might be due to the pbrAP’s weak promoter strength in E. coli. 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.