View source for Part:BBa K1758353

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<partinfo>BBa_K1758353 short</partinfo>

nickel repressor under teh control of a constitutive promoter with the induceble promoter a 5´untranslated region infront of the sfGFP which is used for detection.

===Usage and Biology===
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 <p> Our Nickel biosensor consists of parts of the rcn-operon from <i> E. coli </i> which codes for 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(II)-ions present  the repression of the promoter RcnA will be reversed, because the repressor RcnR binds nickel-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 showed in <Our biosensors >. 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 RcnA 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></html>

<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K1758353 SequenceAndFeatures</partinfo>


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===Functional Parameters===
<partinfo>BBa_K1758353 parameters</partinfo>
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===Results===
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<h2><i>in vivo</i></h2>

<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>

 
 <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 1. 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>
 
 <figure> <img src="https://static.igem.org/mediawiki/2015/8/8e/Bielefeld-CebiTec_in_vivo_Nickel.jpeg" width ="50%" ></a>
<figcaption>Figure 1: 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="col-md-6 text-center" style="margin-bottom: 50px"><figure> 
<img src="https://static.igem.org/mediawiki/2015/c/c9/Bielefeld-CeBiTec_Biolector_nickel.jpg" alt="Adjusting the detection limit" width = "90%">
<figcaption>Figure 2: 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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 <div class="col-md-6 text-center" style="margin-bottom: 50px"><figure><img src="https://static.igem.org/mediawiki/2015/4/41/Bielefeld-CeBiTec_Biolector_nickel_Balkendiagramm.jpeg" width = "90%"></a>
<figcaption>Figure 3: 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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<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 2. 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 2 are represented as bars in figure 3. A fluorescence level difference for 60 min, 150 min and 650 min is represented.</p>

<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 2 and 3). 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 <i>in vitro</i> data using CFPS were taken.</p></br>

<h2>To summarize</h2>
<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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