Difference between revisions of "Part:BBa K1758350"
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===Results===
 
===Results===
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<html>
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<h2><i>in vivo</i></h2>
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<p>We aimed to construct a sensor for nickel detection. It consists of <i>rcnR</i> the repressor and the nickel specific promoter <i>prcnA</i>. The promoter is regulated by the RcnR, which binds Ni<sup>2+</sup>-ions. As the former sensors this one encloses a sfGFP for detection via fluorescence.</p>
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<p> Our nickel biosensor consists of parts of the <i>rcn</i>-operon from E. coli, which encodes a nickel- and cobalt-efflux system. This system is highly sensitive to nickel. In absence of nickel or cobalt RcnR binds to the operator and inhibits the nickel responsive promoter. With Ni<sup>2+</sup>-ions present the repression of the promoter <i>prcnA</i> will be reversed, because the repressor RcnR binds Ni<sup>2+</sup>-ions and cannot attach to the DNA. For our biosensor we construct the part <a href="https://parts.igem.org/Part:BBa_K1758353" target="_blank"> BBa_K1758353 </a> by using the basic construction shown in figure 2. For this part we used the repressor RcnR under control of a constitutive promoter (<a href="https://parts.igem.org/Part:BBa_K1758350" target="_blank"> BBa_K1758350 </a>) and the nickel specific promoter PrcnA with a 5’UTR  in front of sfGFP (<a href="https://parts.igem.org/Part:BBa_K1758352" target="_blank"> BBa_K1758352 </a>) as reporter protein. </p>
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<figure style="width: 600px">
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<a href="https://static.igem.org/mediawiki/2015/8/8e/Bielefeld-CebiTec_in_vivo_Nickel.jpeg" data-lightbox="heavymetals" data-title=" Figure 2: The concept of our <i>in vivo</i> nickel sensor (<a href="https://parts.igem.org/Part:BBa_K1758354" target="_blank"> BBa_K1758354</a>), which consists of the activator under the control of a constitutive promoter (<a href="https://parts.igem.org/Part:BBa_K1758350" target="_blank"> BBa_K1758350</a>)and the operator and promoter sequence of the nickel 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_K1758352" target="_blank"> BBa_K1758352</a>) "><img src="https://static.igem.org/mediawiki/2015/8/8e/Bielefeld-CebiTec_in_vivo_Nickel.jpeg"></a>
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<figcaption>Figure 2: The concept of our <i>in vivo</i> nickel sensor (<a href="https://parts.igem.org/Part:BBa_K1758354" target="_blank"> BBa_K1758354</a>), which consists of the activator under the control of a constitutive promoter (<a href="https://parts.igem.org/Part:BBa_K1758350" target="_blank"> BBa_K1758350</a>)and the operator and promoter sequence of the nickel 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_K1758352" target="_blank"> BBa_K1758352</a>) </figcaption> </figure>
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<div class="row">
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    <div class="col-md-6 text-center" style="margin-bottom: 50px"><figure style="width: 600px">
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<a href="https://static.igem.org/mediawiki/2015/c/c9/Bielefeld-CeBiTec_Biolector_nickel.jpg" data-lightbox="heavymetals" data-title="Figure 3: Time course of the induction of a nickel biosensor with sfGFP for different nickel concentrations in vivo. The data are measured with BioLector and normalized on OD<sub>600</sub>. Error bars represent the standard deviation of two biological replicates.  "><img src="https://static.igem.org/mediawiki/2015/c/c9/Bielefeld-CeBiTec_Biolector_nickel.jpg" alt="Adjusting the detection limit"></a>
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<figcaption>Figure 3: Time course of the induction of a nickel biosensor with sfGFP for different nickel concentrations in vivo. The data are measured with BioLector and normalized on OD<sub>600</sub>. Error bars represent the standard deviation of two biological replicates. </figcaption>
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</figure>
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</div>
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<div class="col-md-6 text-center" style="margin-bottom: 50px"><figure style="width: 600px">
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<a href="https://static.igem.org/mediawiki/2015/4/41/Bielefeld-CeBiTec_Biolector_nickel_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 three biological replicates. "><img src="https://static.igem.org/mediawiki/2015/4/41/Bielefeld-CeBiTec_Biolector_nickel_Balkendiagramm.jpeg" alt="Adjusting the detection limit"></a>
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<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 three biological replicates. </figcaption>
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</figure>
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</div>
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</div>
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<p>We tested our nickel sensor with sfGFP as reporter gene, to test the functionality of the system. Moreover we tested different concentrations. The kinetic of our sensors response to different nickel concentrations is shown in figure 3. The first five hours show a strong decrease in fluorescence. After that there is a slight increase in fluorescence. Starting levels of fluorescence are not reached. 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>
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<p> The data for our nickel sensor show a trend that differs for that of the other sensors. There is no indication for a working sensor <i>in vivo</i> (Figure 3 and 4). There is a fluorescence signal, but it decreases in the first five hours. After reaching a minimum the fluorescence increases slowly. Additionally, there is no difference in fluorescence as response to various nickel concentration. Nickel could influence the cells and thereby caused a precipitation, which could result in decrease of fluorescence.
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With this sensor no production of sfGFP via fluorescence level change could be detected. Therefore, this sensor is not suitable for our approach. Due to this observation, no <i>in vitro</i> data using CFPS were taken.</p></br>
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<h2>To summarize</h2>
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<p>With this sensor no production of sfGFP via fluorescence level change could be detected. Therefore, this sensor is not suitable for our approach. Consequently, no <i>in vitro</i> tests were performed.
 +
To create a working sensor based on this concept further optimization is needed.</p>
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</div>
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</html>

Revision as of 05:57, 19 September 2015

Nickel repressor under control of constitutive promoter and strong RBS

nickel repressor under the control of a constitutive promoter.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

in vivo

We aimed to construct a sensor for nickel detection. It consists of rcnR the repressor and the nickel specific promoter prcnA. The promoter is regulated by the RcnR, which binds Ni2+-ions. As the former sensors this one encloses a sfGFP for detection via fluorescence.

Our nickel biosensor consists of parts of the rcn-operon from E. coli, which encodes a nickel- and cobalt-efflux system. This system is highly sensitive to nickel. In absence of nickel or cobalt RcnR binds to the operator and inhibits the nickel responsive promoter. With Ni2+-ions present the repression of the promoter prcnA will be reversed, because the repressor RcnR binds Ni2+-ions and cannot attach to the DNA. For our biosensor we construct the part BBa_K1758353 by using the basic construction shown in figure 2. For this part we used the repressor RcnR under control of a constitutive promoter ( BBa_K1758350 ) and the nickel specific promoter PrcnA with a 5’UTR in front of sfGFP ( BBa_K1758352 ) as reporter protein.

BBa_K1758354), which consists of the activator under the control of a constitutive promoter ( BBa_K1758350)and the operator and promoter sequence of the nickel inducible promoter. An untranslated region in front of the sfGFP, which is used for detection, enhances its expression ( BBa_K1758352) ">
Figure 2: The concept of our in vivo nickel sensor ( BBa_K1758354), which consists of the activator under the control of a constitutive promoter ( BBa_K1758350)and the operator and promoter sequence of the nickel inducible promoter. An untranslated region in front of the sfGFP, which is used for detection, enhances its expression ( BBa_K1758352)
Adjusting the detection limit
Figure 3: Time course of the induction of a nickel biosensor with sfGFP for different nickel concentrations in vivo. The data are measured with BioLector and normalized on OD600. Error bars represent the standard deviation of two biological replicates.
Adjusting the detection limit
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 three biological replicates.

We tested our nickel sensor with sfGFP as reporter gene, to test the functionality of the system. Moreover we tested different concentrations. The kinetic of our sensors response to different nickel concentrations is shown in figure 3. The first five hours show a strong decrease in fluorescence. After that there is a slight increase in fluorescence. Starting levels of fluorescence are not reached. 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 data for our nickel sensor show a trend that differs for that of the other sensors. There is no indication for a working sensor in vivo (Figure 3 and 4). There is a fluorescence signal, but it decreases in the first five hours. After reaching a minimum the fluorescence increases slowly. Additionally, there is no difference in fluorescence as response to various nickel concentration. Nickel could influence the cells and thereby caused a precipitation, which could result in decrease of fluorescence. With this sensor no production of sfGFP via fluorescence level change could be detected. Therefore, this sensor is not suitable for our approach. Due to this observation, no in vitro data using CFPS were taken.


To summarize

With this sensor no production of sfGFP via fluorescence level change could be detected. Therefore, this sensor is not suitable for our approach. Consequently, no in vitro tests were performed. To create a working sensor based on this concept further optimization is needed.