Difference between revisions of "Team:Bielefeld-CeBiTec/Results/HeavyMetals"

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<h2><i>in vivo</i></h2></br></br>  
 
<h2><i>in vivo</i></h2></br></br>  
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Our sensor for copper detection consists of CueR a MerR like activator and the copper specific promoter CopAP. The promoter is regulated by the CueR, which binds Cu-ions. We also used a sfGFP behind the promoter for detection trough a fluorescence signal. </br></br>   
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Our sensor for copper detection consists of CueR a MerR like activator and the copper specific promoter CopAP. The promoter is regulated by CueR, which binds Cu2+-ions. We also used a sfGFP behind the promoter for detection trough a fluorescence signal. </br></br>   
 
<figure style="width: 600px">
 
<figure style="width: 600px">
 
<a href="https://static.igem.org/mediawiki/2015/9/90/Bielefeld-CeBiTec_Biolector_copper.jpg" data-lightbox="heavymetals" data-title="Time course of the induction of a chromium biosensor with sfGFP for different chromium concentrations in vivo. The data are measured with BioLector and normalized on OD600. Error bars represent the standard deviation of two biological replicates."><img src="https://static.igem.org/mediawiki/2015/9/90/Bielefeld-CeBiTec_Biolector_copper.jpg" alt="Adjusting the detection limit"></a>
 
<a href="https://static.igem.org/mediawiki/2015/9/90/Bielefeld-CeBiTec_Biolector_copper.jpg" data-lightbox="heavymetals" data-title="Time course of the induction of a chromium biosensor with sfGFP for different chromium concentrations in vivo. The data are measured with BioLector and normalized on OD600. Error bars represent the standard deviation of two biological replicates."><img src="https://static.igem.org/mediawiki/2015/9/90/Bielefeld-CeBiTec_Biolector_copper.jpg" alt="Adjusting the detection limit"></a>
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<figure style="width: 600px">
 
<figure style="width: 600px">
 
<a href="http://https://static.igem.org/mediawiki/2015/4/4e/Bielefeld-CeBiTec_Biolector_copper_Balkendiagramm.jpeg" data-lightbox="heavymetals" data-title="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/2015/4/4e/Bielefeld-CeBiTec_Biolector_copper_Balkendiagramm.jpeg" alt="Adjusting the detection limit"></a>
 
<a href="http://https://static.igem.org/mediawiki/2015/4/4e/Bielefeld-CeBiTec_Biolector_copper_Balkendiagramm.jpeg" data-lightbox="heavymetals" data-title="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/2015/4/4e/Bielefeld-CeBiTec_Biolector_copper_Balkendiagramm.jpeg" alt="Adjusting the detection limit"></a>
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<figcaption>Fluorescence levels at three different stages of cultivation. Shown are levels after 60 minutes, 150 minutes and 650 minutes. .</figcaption>
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<figcaption>Fluorescence levels at three different stages of cultivation. Shown are levels after 60 minutes, 150 minutes and 650 minutes.</figcaption>
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</figure>
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</figure></br>
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The shown data suggest that sensing copper with our device is possible even if the detectable concentrations are higher than the desireble sensitivity limits. Therfore we tested the copper sensor in our <i>in vitro</i> transcription translation approach. 
 
<h2><i>in vitro</i></h2>
 
<h2><i>in vitro</i></h2>
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</br></br>
 
<figure style="width: 600px">
 
<figure style="width: 600px">
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<a href="https://static.igem.org/mediawiki/2015/3/37/Bielefeld-CeBiTec_Influence_of_copper_on_the_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/2015/3/37/Bielefeld-CeBiTec_Influence_of_copper_on_the_cell_extract.jpeg" alt="Adjusting the detection limit"></a>
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<a href="https://static.igem.org/mediawiki/2015/3/37/Bielefeld-CeBiTec_Influence_of_copper_on_the_cell_extract.jpeg" data-lightbox="heavymetals" data-title="Influence of different copper concentrations on our crude cell extract. Error bars represent the standard deviation of three biological replicates."><img src="https://static.igem.org/mediawiki/2015/3/37/Bielefeld-CeBiTec_Influence_of_copper_on_the_cell_extract.jpeg" alt="Adjusting the detection limit"></a>
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<figcaption>TEXT. Error bars represent the standard deviation of three biological replicates.</figcaption>
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<figcaption>Influence of different copper concentrations on our crude cell extract. Error bars represent the standard deviation of three biological replicates.</figcaption>
 
</figure>
 
</figure>
  
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<figure style="width: 600px">
 
<figure style="width: 600px">
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<a href="https://static.igem.org/mediawiki/2015/4/45/Bielefeld-CeBiTec_induction_copper_in_CueR_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/2015/4/45/Bielefeld-CeBiTec_induction_copper_in_CueR_cell-extract.jpeg" alt="Adjusting the detection limit"></a>
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<a href="https://static.igem.org/mediawiki/2015/4/45/Bielefeld-CeBiTec_induction_copper_in_CueR_cell-extract.jpeg" data-lightbox="heavymetals" data-title="Copper specific cell extract made from <i>E. coli</i> cells which have already expressed the activator before cell extract production. Induction of copper induceble promoter without T7 infont of the operator site with different copper concentrations. Error bars represent the standard deviation of three biological replicates."><img src="https://static.igem.org/mediawiki/2015/4/45/Bielefeld-CeBiTec_induction_copper_in_CueR_cell-extract.jpeg" alt="Adjusting the detection limit"></a>
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<figcaption>TEXT. Error bars represent the standard deviation of three biological replicates.</figcaption>
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<figcaption>Copper specific cell extract made from <i>E. coli</i> cells which have already expressed the activator before cell extract production. Induction of copper induceble promoter without T7 infont of the operator site with different copper concentrations. Error bars represent the standard deviation of three biological replicates.</figcaption>
 
</figure>
 
</figure>
  
 
<figure style="width: 600px">
 
<figure style="width: 600px">
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<a href="https://static.igem.org/mediawiki/2015/4/4c/Bielefeld-CeBiTec_correction_induction_copper_in_cueR_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/2015/4/4c/Bielefeld-CeBiTec_correction_induction_copper_in_cueR_cell-extract.jpeg" alt="Adjusting the detection limit"></a>
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<a href="https://static.igem.org/mediawiki/2015/4/4c/Bielefeld-CeBiTec_correction_induction_copper_in_cueR_cell-extract.jpeg" data-lightbox="heavymetals" data-title="Copper specific cell extract made from <i>E. coli</i> cells which have already expressed the activator before cell extract production. Induction of copper inducible promoter without T7 in front of the operator site with different copper concentrations. Error bars represent the standard deviation of three biological replicates. Data are normalized on coppers influence to the cell extract."><img src="https://static.igem.org/mediawiki/2015/4/4c/Bielefeld-CeBiTec_correction_induction_copper_in_cueR_cell-extract.jpeg" alt="Adjusting the detection limit"></a>
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<figcaption>TEXT. Error bars represent the standard deviation of three biological replicates.</figcaption>
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<figcaption>Copper specific cell extract made from <i>E. coli</i> cells which have already expressed the activator before cell extract production. Induction of copper inducible promoter without T7 in front of the operator site with different copper concentrations. Error bars represent the standard deviation of three biological replicates. Data are normalized on coppers influence to the cell extract.</figcaption>
 
</figure>
 
</figure>
  
 
<figure style="width: 600px">
 
<figure style="width: 600px">
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<a href="https://static.igem.org/mediawiki/2015/c/ce/Bielefeld-CeBiTec_induction_T7-copAP_copper_in_cueR_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/c/ce/Bielefeld-CeBiTec_induction_T7-copAP_copper_in_cueR_cell-extract.jpeg" alt="Adjusting the detection limit"></a>
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<a href="https://static.igem.org/mediawiki/2015/c/ce/Bielefeld-CeBiTec_induction_T7-copAP_copper_in_cueR_cell-extract.jpeg" data-lightbox="heavymetals" data-title="Copper specific cell extract made from <i>E. coli</i> cells which have already expressed the activator before cell extract production. Induction with different copper concentrations. Error bars represent the standard deviation of three biological replicates."><img src="https://static.igem.org/mediawiki/2015/c/ce/Bielefeld-CeBiTec_induction_T7-copAP_copper_in_cueR_cell-extract.jpeg" alt="Adjusting the detection limit"></a>
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<figcaption>TEXT. Error bars represent the standard deviation of three biological replicates.</figcaption>
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<figcaption>Copper specific cell extract made from <i>E. coli</i> cells which have already expressed the activator before cell extract production. Induction with different copper concentrations. Error bars represent the standard deviation of three biological replicates.</figcaption>
 
</figure>
 
</figure>
  

Revision as of 05:01, 14 September 2015

iGEM Bielefeld 2015


Heavy Metals

Zusammenfassung in ganz wenigen Worten.


The different sensors we worked with were characterized in vivo as well as in vitro.

We tested the influence of each heavy metal on our sensors in vivo Therefore we used heavy metal concentrations based on heavy metal occurrences measured all over the world.
Adjusting the detection limit
Influence of heavy metals on the growth of E.coli KRX shown is the standard deviation of three biological replicates. For induction concentrations of 20 µg/L lead, 60 µg/L mercury, 60 µg/L chromium, 80 µg/L nickel, 40 mg/L copper which represent ten times of the WHO guideline were used.



The tested heavy metal concentrations had no negative effect on E. colis growth. Moreover there is no significant difference between the curves with heavy metals and the controls. This first experiment showed us, in vivo characterization with these sensors under the tested heavy metal concentrations is possible. Most of our sensors were cultivated in the BioLector. Due to the accuracy of this device we could measure our sample in duplicates.

Arsenic

in vivo

Adjusting the detection limit
Time course of the induction of an arsenic biosensor with RFP for different arsenic concentrations in vivo. Error bars represent the standard deviation of three biological replicates.

in vitro

Chromium



in vivo



Our sensor for chromium detection consists of ChrB the repressor and the chromate specific promoter ChrP. The promoter is regulated by the ChrB, which binds Cr-ions. Behind the promoter is a sfGFP for detection of a fluorescence signal. In vivo we could show that the addition of different concentrations of chromium have different effects to transcription of sfGFP.

Adjusting the detection limit
Time course of the induction of a chromium biosensor with sfGFP for different chromium 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
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.

Our data lead to the conclusion that in a cell based system it is possible to detect chromium. In contrast to our expectations with higher chromium concentrations we got lower fluorescence levels. These observations needed further investigation.

in vitro

Adjusting the detection limit
Influence of different chromium concentrations on our crude cell extract. Error bars represent the standard deviation of three biological replicates.
Adjusting the detection limit
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.
Adjusting the detection limit
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.Data are normalised on chromiums influence to the cell extrat.
Adjusting the detection limit
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 withdifferent chromium concentrations. Error bars represent the standard deviation of three biological replicates.
Adjusting the detection limit
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 withdifferent chromium concentrations. Error bars represent the standard deviation of three biological replicates.Data are normalised on chromiums influence to the cell extrat.

Copper

in vivo



Our sensor for copper detection consists of CueR a MerR like activator and the copper specific promoter CopAP. The promoter is regulated by CueR, which binds Cu2+-ions. We also used a sfGFP behind the promoter for detection trough a fluorescence signal.

Adjusting the detection limit
Time course of the induction of a chromium biosensor with sfGFP for different chromium concentrations in vivo. The data are measured with BioLector and normalized on OD600. Error bars represent the standard deviation of two biological replicates.


In vivo we could show that the adding different concentrations of copper has different effects on the transcription of sfGFP.

Adjusting the detection limit
Fluorescence levels at three different stages of cultivation. Shown are levels after 60 minutes, 150 minutes and 650 minutes.

The shown data suggest that sensing copper with our device is possible even if the detectable concentrations are higher than the desireble sensitivity limits. Therfore we tested the copper sensor in our in vitro transcription translation approach.

in vitro



Adjusting the detection limit
Influence of different copper concentrations on our crude cell extract. Error bars represent the standard deviation of three biological replicates.
Adjusting the detection limit
TEXT. Error bars represent the standard deviation of three biological replicates.
Adjusting the detection limit
Copper specific cell extract made from E. coli cells which have already expressed the activator before cell extract production. Induction of copper induceble promoter without T7 infont of the operator site with different copper concentrations. Error bars represent the standard deviation of three biological replicates.
Adjusting the detection limit
Copper specific cell extract made from E. coli cells which have already expressed the activator before cell extract production. Induction of copper inducible promoter without T7 in front of the operator site with different copper concentrations. Error bars represent the standard deviation of three biological replicates. Data are normalized on coppers influence to the cell extract.
Adjusting the detection limit
Copper specific cell extract made from E. coli cells which have already expressed the activator before cell extract production. Induction with different copper concentrations. Error bars represent the standard deviation of three biological replicates.
Adjusting the detection limit
TEXT. Error bars represent the standard deviation of three biological replicates.

Lead

in vivo

Adjusting the detection limit
TEXT. Error bars represent the standard deviation of three biological replicates.
Adjusting the detection limit
TEXT. Error bars represent the standard deviation of three biological replicates.

Mercury

in vivo

Adjusting the detection limit
TEXT. Error bars represent the standard deviation of three biological replicates.
Adjusting the detection limit
TEXT. Error bars represent the standard deviation of three biological replicates.

in vitro

Adjusting the detection limit
TEXT. Error bars represent the standard deviation of three biological replicates.
Adjusting the detection limit
TEXT. Error bars represent the standard deviation of three biological replicates.
Adjusting the detection limit
TEXT. Error bars represent the standard deviation of three biological replicates.
Adjusting the detection limit
TEXT. Error bars represent the standard deviation of three biological replicates.

Nickel

in vivo

Adjusting the detection limit
TEXT. Error bars represent the standard deviation of three biological replicates.
Adjusting the detection limit
TEXT. Error bars represent the standard deviation of three biological replicates.

And the date rape drugs?