Difference between revisions of "Part:BBa K1758314"
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<partinfo>BBa_K1758320 short</partinfo></br>
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<partinfo>BBa_K1758314 short</partinfo>
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Chromium induceble promoter under the control of a T7 promoter with 5´untranslated region and sfGFP for detection
 
Chromium induceble promoter under the control of a T7 promoter with 5´untranslated region and sfGFP for detection
  
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<html>
 
<html>
 
This part is essential for our <i> in vitro </i> characterization of our chromium sensor.
 
This part is essential for our <i> in vitro </i> characterization of our chromium sensor.
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The edition of T7 promoter to <a href=https://parts.igem.org/Part:BBa_K1758313"target="_blank">BBa_K1758313</a> enables us to characterize tihs sensor in our <a href=http://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS"target="_blank">CFPS</a>.</br>
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The edition of T7 promoter to <a href=https://parts.igem.org/Part:BBa_K1758313"target="_blank">BBa_K1758313</a> enables us to characterize this sensor in our <a href=http://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS"target="_blank">CFPS</a>.</br>
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We used this promoter for our chromium sensor. Originaly its from <i>Ochrobactrum triti</i> ci5bvl1.
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</html>this part cerates our chromium sensor.
 
</html>
 
</html>
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1758320 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K1758314 SequenceAndFeatures</partinfo>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K1758320 parameters</partinfo>
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<partinfo>BBa_K1758314 parameters</partinfo>
 
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<h2><i>in vitro</i></h2>
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<p>For the characterization of the chromium sensor with CFPS we used parts differing from that we used in vivo characterization. For the in vitro characterization we used a cell extract produced from cells which contain the plasmid (<a href="https://parts.igem.org/Part:BBa_K1758310" target="_blank">BBa_K1758310</a>). The plasmid contains the gene <i>chrB</i> under the control of a constitutive promoter, so that the cell extract is enriched with repressor molecules. In addition to that we added plasmid-DNA of the chromium specific promoter <i>chrP</i> with 5’UTR-sfGFP under the control of T7-promoter (<a href="https://parts.igem.org/Part:BBa_K1758314" target="_blank">BBa_K1758314</a> (figure 6))to the cell extract. The T7-promoter is needed to get a better fluorescence expression. </p>   
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<figure style="float:left; width:30%;">
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  <img src=" https://static.igem.org/mediawiki/2015/e/e4/Bielefeld-CeBiTec_in_vitro_ChrB-part.jpeg" width="100%" alt="repressor construct used for in vivo characterisation"><figcaption> Figure 5: To produce the cell extract for <i>in vitro</i> characterization a construct (<a href="https://parts.igem.org/Part:BBa_K1758310" target="_blank">BBa_K1758310</a> ) with chromium repressor under the control of a constitutive promoter and strong RBS (BBa_K608002)  is needed.</figcaption>
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</figure>
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    <figure style="float:right; width:30%;">
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  <img src=" https://static.igem.org/mediawiki/2015/1/1f/Bielefeld-CebiTec_in_vitro_T7-chrP-UTR-sfGFP.jpeg" width="100%" alt="promoter construct used for in vivo characterisation "><figcaption> T7-chrP-UTR-sfGFP <a href="https://parts.igem.org/Part:BBa_K1758314" target="_blank">BBa_K1758314</a> used for<i>in vitro</i> characterization.</figcaption>
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</figure> 
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<!--Chrom bei niedrigeren Konzentrationen keinen signifikaten einfluss auf das System selbst, erst bei hoher Konzentration zeigt sich das CFPS nicht mehr in der lage ist ein Fluorescence signal zu erzeugen, zeigt, dass einen zu hohe konzentration das System zerstört. die Translations bzw Transskriptions ... sind nicht mehr in der Lage zu arbeiten, also keine -->
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<figure  width="50%">
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<img src="https://static.igem.org/mediawiki/2015/9/99/Bielefeld-CeBiTec_Influence_of_chromium_on_the_cell_extract.jpeg" alt="Adjusting the detection limit" width="100%">
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<figcaption>Figure 7: Influence of different chromium concentrations on our crude cell extract. Error bars represent the standard deviation of three biological replicates. </figcaption>
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</figure>
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<p>Chromium’s influence on the cell extract as shown in figure 7 is minimal for low concentrations. Higher chromium concentrations have a measurable impact on the viability of the cell extract, which is visible at concentrations of 120 µg/L and obvious at concentrations of 240 µg/L chromium.</p>
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<figure width="50%">
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<img src="https://static.igem.org/mediawiki/2015/f/fb/Bielefeld-CeBiTec_induction_chromium_in_chrB_cell_extract.jpg" alt="Adjusting the detection limit" width="100%">
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<figcaption>Figure 8: Chromium specific cell extract made from <i>E. coli</i> cells which already expressed the repressor before cell extract production. Induction with different chromium concentrations. Error bars represent the standard deviation of three biological replicates. </figcaption>
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</figure>
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<p>The decrease of fluorescence for higher chromium concentrations in chromium specific cell extract is shown in figure 8. An increase of fluorescence at higher chromium concentrations would have been expected resulting out of the induction of the chromium sensor.
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A factor which should be considered is the influence of high chromium concentrations to the cell extract. The test for influence of chromium on the specific cell extract, illustrated in figure 7 showed that the influence of chromium at low concentrations is not significant. But the graphic shows that high concentrations of chromium induce fatal damages to the cell extract. </p>
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  <figure width="50%">
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<img src="https://static.igem.org/mediawiki/2015/1/1e/Bielefeld-CeBiTec_correction_induction_chromium_in_chrB-cell-extract.jpeg" alt="Adjusting the detection limit" width="100%">
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<figcaption>Figure 9: Chromium specific cell extract made from <i>E. coli</i> cells which already expressed the repressor before cell extract production. Induction with different chromium concentrations. The data are normalized on chromium’s influence to the cell extract. Error bars represent the standard deviation of three biological replicates. </figcaption>
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</figure>
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<p>Taking the influence of different chromium concentrations under consideration measured fluorescence can be normalized on chromium’s influence on the cell extract (figure 9). Normalized data suggest, that higher concentrations of chromium induce fluorescence in relevance to chromium’s influence on the cell extract. </p>
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<figure width=50%>
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<img src="https://static.igem.org/mediawiki/2015/b/bd/Bielefeld-CeBiTec_induction_chromium_in_chrB_optimized_cell_extract2.jpg" alt="Adjusting the detection limit" width="100%">
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<figcaption>Figure 10: Chromium sensor with alternative repressor build by team Dundee 2015, which has only the first 15 codons optimized in chromium specific cell extract under the induction with different chromium concentrations. Error bars represent the standard deviation of three biological replicates. </figcaption>
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</figure>
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<figure width="50%">
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<img src="https://static.igem.org/mediawiki/2015/f/fe/Bielefeld-CeBiTec_Corr-induction-Cr-in-ChrBopt-CE.jpeg" alt="Adjusting the detection limit" width="100%">
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<figcaption>Figure 11: Chromium sensor with alternative repressor build by team Dundee 2015, which has only the first 15 codons optimized in chromium specific cell extract under the induction with different chromium concentrations. Data are normalized on chromium’s influence to the specific cell extract Error bars represent the standard deviation of three biological replicates. </figcaption>
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</figure>
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<p>In addition to the measurements of our chromium sensor in CFPS we measured our chromium inducible promoter with the repressor of team Dundee (figure 10, 11), which works similar to ours. In contrast to our repressor only first 15 codons of their repressor are codon-optimized. Measurements with their repressor showed tendencies similar to our measured repressor. After normalization induction with higher chromium concentrations showed a detectable fluorescence response for both measured datasets. </p>
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</p>
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</html>

Latest revision as of 12:16, 20 September 2015

Chromium responsive promoter under T7-promoter with UTR-sfGFP

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

Usage and Biology

This part is essential for our in vitro characterization of our chromium sensor. The edition of T7 promoter to BBa_K1758313 enables us to characterize this sensor in our CFPS.
We used this promoter for our chromium sensor. Originaly its from Ochrobactrum triti ci5bvl1. this part cerates our chromium sensor. </html> Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 167
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 260



in vitro

For the characterization of the chromium sensor with CFPS we used parts differing from that we used in vivo characterization. For the in vitro characterization we used a cell extract produced from cells which contain the plasmid (BBa_K1758310). The plasmid contains the gene chrB under the control of a constitutive promoter, so that the cell extract is enriched with repressor molecules. In addition to that we added plasmid-DNA of the chromium specific promoter chrP with 5’UTR-sfGFP under the control of T7-promoter (BBa_K1758314 (figure 6))to the cell extract. The T7-promoter is needed to get a better fluorescence expression.

repressor construct used for in vivo characterisation
Figure 5: To produce the cell extract for in vitro characterization a construct (BBa_K1758310 ) 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-chrP-UTR-sfGFP BBa_K1758314 used forin vitro characterization.
Adjusting the detection limit
Figure 7: Influence of different chromium concentrations on our crude cell extract. Error bars represent the standard deviation of three biological replicates.

Chromium’s influence on the cell extract as shown in figure 7 is minimal for low concentrations. Higher chromium concentrations have a measurable impact on the viability of the cell extract, which is visible at concentrations of 120 µg/L and obvious at concentrations of 240 µg/L chromium.

Adjusting the detection limit
Figure 8: Chromium specific cell extract made from E. coli cells which already expressed the repressor before cell extract production. Induction with different chromium concentrations. Error bars represent the standard deviation of three biological replicates.

The decrease of fluorescence for higher chromium concentrations in chromium specific cell extract is shown in figure 8. An increase of fluorescence at higher chromium concentrations would have been expected resulting out of the induction of the chromium sensor. A factor which should be considered is the influence of high chromium concentrations to the cell extract. The test for influence of chromium on the specific cell extract, illustrated in figure 7 showed that the influence of chromium at low concentrations is not significant. But the graphic shows that high concentrations of chromium induce fatal damages to the cell extract.

Adjusting the detection limit
Figure 9: Chromium specific cell extract made from E. coli cells which already expressed the repressor before cell extract production. Induction with different chromium concentrations. The data are normalized on chromium’s influence to the cell extract. Error bars represent the standard deviation of three biological replicates.

Taking the influence of different chromium concentrations under consideration measured fluorescence can be normalized on chromium’s influence on the cell extract (figure 9). Normalized data suggest, that higher concentrations of chromium induce fluorescence in relevance to chromium’s influence on the cell extract.

Adjusting the detection limit
Figure 10: Chromium sensor with alternative repressor build by team Dundee 2015, which has only the first 15 codons optimized in chromium specific cell extract under the induction with different chromium concentrations. Error bars represent the standard deviation of three biological replicates.
Adjusting the detection limit
Figure 11: Chromium sensor with alternative repressor build by team Dundee 2015, which has only the first 15 codons optimized in chromium specific cell extract under the induction with different chromium concentrations. Data are normalized on chromium’s influence to the specific cell extract Error bars represent the standard deviation of three biological replicates.

In addition to the measurements of our chromium sensor in CFPS we measured our chromium inducible promoter with the repressor of team Dundee (figure 10, 11), which works similar to ours. In contrast to our repressor only first 15 codons of their repressor are codon-optimized. Measurements with their repressor showed tendencies similar to our measured repressor. After normalization induction with higher chromium concentrations showed a detectable fluorescence response for both measured datasets.