Difference between revisions of "Part:BBa K1758344"

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<partinfo>BBa_K1758344 parameters</partinfo>
 
<partinfo>BBa_K1758344 parameters</partinfo>
 
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<h2><i>in vitro</i></h2>
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<p>For the characterization of the mercury sensor with CFPS we used parts differing from that we used in the <i>in vivo</i> characterization. For the <i>in vitro</i> characterization we used a cell extract out of cells, which contained the plasmid (<a href="https://parts.igem.org/Part:BBa_K1758340" target="_blank"> BBa_K1758340</a>)(figure 5). In addition, we added plasmid DNA to the cell extract. This plasmid consisted of the mercury specific promoter <i>pmerT</i> with 5’-UTR-sfGFP. The entire sequence was placed under the control of of T7-promoter (<a href="https://parts.igem.org/Part:BBa_K1758344" target="_blank"> BBa_K1758344</a>)(figure 6). The T7-promoter is needed to get a better fluorescence expression.</p>   
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    <div class="col-md-6 text-center" style="margin-bottom: 50px"> <figure style="width: 400px">
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  <a href=" https://static.igem.org/mediawiki/2015/3/3c/Bielefeld-CeBiTec_in_vitro_merR-part.jpeg" data-lightbox="heavymetals" data-title=" Figure 5: To produce the cell extract for <i>in vitro</i> characterization a construct (BBa_K1758340 ) with chromium repressor under the control of a constitutive promoter and strong RBS. " alt="repressor construct used for in vivo characterization."><img src=" https://static.igem.org/mediawiki/2015/3/3c/Bielefeld-CeBiTec_in_vitro_merR-part.jpeg" alt="repressor construct used for in vitro characterisation"></a> <figcaption>Figure 5: To produce the cell extract for <i>in vitro</i> characterization a construct (<a href="https://parts.igem.org/Part:BBa_K175840" target="_blank">BBa_K175840</a>) with chromium repressor under the control of a constitutive promoter and strong RBS (BBa_K608002) is needed. </figcaption>
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    <div class="col-md-6 text-center" style="margin-bottom: 50px"> <figure style="width: 400px">
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  <a href=" https://static.igem.org/mediawiki/2015/e/e2/Bielefeld-CebiTec_in_vitro_T7-merT-UTR-sfGFP.jpeg " data-lightbox="heavymetals" data-title="T7-PmerT-UTR-sfGFP used for<i>in vitro</i> characterization." https://static.igem.org/mediawiki/2015/e/e2/Bielefeld-CebiTec_in_vitro_T7-merT-UTR-sfGFP.jpeg " alt="promoter construct used for in vivo characterization."><img src=" https://static.igem.org/mediawiki/2015/e/e2/Bielefeld-CebiTec_in_vitro_T7-merT-UTR-sfGFP.jpeg" alt="promoter construct used for in vivo characterisation "></a> <figcaption>T7-<i>PmerT</i>-UTR-sfGFP <a href="https://parts.igem.org/Part:BBa_K1758344" target="_blank"> BBa_K175844</a> used for<i>in vitro</i> characterization.</figcaption>
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<a href="https://static.igem.org/mediawiki/2015/b/b9/Bielefeld-CeBiTec_Influence_of_mercury_on_the_cell_extract.jpeg" data-lightbox="heavymetals" data-title="Figure 7: Influence of different mercury concentrations on our crude cell extract. Error bars represent the standard deviation of three biological replicates. "><img src="https://static.igem.org/mediawiki/2015/b/b9/Bielefeld-CeBiTec_Influence_of_mercury_on_the_cell_extract.jpeg" alt="Adjusting the detection limit"></a>
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<figcaption>Figure 7: Influence of different mercury concentrations on our crude cell extract. Error bars represent the standard deviation of three biological replicates.</figcaption>
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<a href="https://static.igem.org/mediawiki/2015/1/1e/Bielefeld-CeBiTec_Abnahme_unter_Hg.jpeg" data-lightbox="heavymetals" data-title="TEXT Error bars represent the standard deviation of three biological replicates."><img src="https://static.igem.org/mediawiki/2015/1/1e/Bielefeld-CeBiTec_Abnahme_unter_Hg.jpeg" alt="Adjusting the detection limit"></a>
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<figcaption>Influence of different mercury concentrations on our crude cell extract. Error bars represent the standard deviation of three biological replicates.</figcaption>
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<a href="https://static.igem.org/mediawiki/2015/7/7f/Bielefeld-CeBiTec_induction_mercury_in_merR_cell-extract.jpeg" data-lightbox="heavymetals" data-title="Figure 8: Mercury specific cell extract made from E. coli cells, which have already expressed the activator before cell extract production. Induction of mercury inducible promoter without T7 in front of the operator site with different mercury concentrations. Error bars represent the standard deviation of three biological replicates. "><img src="https://static.igem.org/mediawiki/2015/7/7f/Bielefeld-CeBiTec_induction_mercury_in_merR_cell-extract.jpeg" alt="Adjusting the detection limit"></a>
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<figcaption>Figure 8: Mercury specific cell extract made from E. coli cells, which have already expressed the activator before cell extract production. Induction of mercury inducible promoter without T7 in front of the operator site with different mercury concentrations. Error bars represent the standard deviation of three biological replicates. </figcaption>
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<a href="https://static.igem.org/mediawiki/2015/f/f9/Bielefeld-CeBiTec_correction_induction_mercury_in_merR_cell-extract.jpeg" data-lightbox="heavymetals" data-title="TEXT Error bars represent the standard deviation of three biological replicates."><img src="https://static.igem.org/mediawiki/2015/f/f9/Bielefeld-CeBiTec_correction_induction_mercury_in_merR_cell-extract.jpeg" alt="Adjusting the detection limit"></a>
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<figcaption>Figure 9: Mercury specific cell extract made from <i>E. coli</i> cells, which have already expressed the activator before cell extract production. Induction of mercury inducible promoter without T7 in front of the operator site with different mercury concentrations. Error bars represent the standard deviation of three biological replicates. </figcaption>
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<p><i>In vitro</i> this sensor showed good results. The fluorescence level was high at low concentrations. Additionally, it showed that the expression level at 6 µg/L (Guideline of WHO for Mercury) reached the maximal signal. This result indicated the potential for measurement of concentrations under 6 µg/L.To confirm this hypothesis, it takes more experiments and tests with lower concentrations. Due to the high expression of sfGFP at low concentrations and the same expression level at different concentrations, it is not possible to quantify mercury with CFPS analyses . , Our model predicted this observation. During the measurement we noticed that the heavy metals have negative influences on the cell extract. Because of this fact, we used a correction factor, which resulted from the heavy metals influence on the CFPS system. This already optimized sensor showed the high potential of optimized sensors in CFPS.</p>
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Revision as of 03:38, 19 September 2015

Mercury responsive promoter with T7-promoter and UTR-sfGFP

mercury induceble promoter under the control of a T7 promoter with 5´untranslated region and sfGFP for detection

Usage and Biology

For the in vitro characterization we used a cell extract out of cells which contain the Plasmid ( BBa_K1758340). In addition to that we added Plasmid-DNA of the copper specific promoter merT with 5’UTR-sfGFP under the control of T7-promoter ( BBa_K1758344)to the cell extract. The T7-promoter is needed to get a better fluorescence expression.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 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 145


in vitro

For the characterization of the mercury sensor with CFPS we used parts differing from that we used in the in vivo characterization. For the in vitro characterization we used a cell extract out of cells, which contained the plasmid ( BBa_K1758340)(figure 5). In addition, we added plasmid DNA to the cell extract. This plasmid consisted of the mercury specific promoter pmerT with 5’-UTR-sfGFP. The entire sequence was placed under the control of of T7-promoter ( BBa_K1758344)(figure 6). The T7-promoter is needed to get a better fluorescence expression.

repressor construct used for in vitro characterisation
Figure 5: To produce the cell extract for in vitro characterization a construct (BBa_K175840) with chromium repressor under the control of a constitutive promoter and strong RBS (BBa_K608002) is needed.
promoter construct used for in vivo characterisation
T7-PmerT-UTR-sfGFP BBa_K175844 used forin vitro characterization.
Adjusting the detection limit
Figure 7: Influence of different mercury concentrations on our crude cell extract. Error bars represent the standard deviation of three biological replicates.
Adjusting the detection limit
Figure 8: Mercury specific cell extract made from E. coli cells, which have already expressed the activator before cell extract production. Induction of mercury inducible promoter without T7 in front of the operator site with different mercury concentrations. Error bars represent the standard deviation of three biological replicates.
Adjusting the detection limit
Figure 9: Mercury specific cell extract made from E. coli cells, which have already expressed the activator before cell extract production. Induction of mercury inducible promoter without T7 in front of the operator site with different mercury concentrations. Error bars represent the standard deviation of three biological replicates.

In vitro this sensor showed good results. The fluorescence level was high at low concentrations. Additionally, it showed that the expression level at 6 µg/L (Guideline of WHO for Mercury) reached the maximal signal. This result indicated the potential for measurement of concentrations under 6 µg/L.To confirm this hypothesis, it takes more experiments and tests with lower concentrations. Due to the high expression of sfGFP at low concentrations and the same expression level at different concentrations, it is not possible to quantify mercury with CFPS analyses . , Our model predicted this observation. During the measurement we noticed that the heavy metals have negative influences on the cell extract. Because of this fact, we used a correction factor, which resulted from the heavy metals influence on the CFPS system. This already optimized sensor showed the high potential of optimized sensors in CFPS.