Difference between revisions of "Team:Bielefeld-CeBiTec/Results/HeavyMetals"
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<a href="https://static.igem.org/mediawiki/2015/a/aa/Bielefeld-CeBiTec_Biolector_lead_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/a/aa/Bielefeld-CeBiTec_Biolector_lead_Balkendiagramm.jpeg" alt="Adjusting the detection limit"></a> | <a href="https://static.igem.org/mediawiki/2015/a/aa/Bielefeld-CeBiTec_Biolector_lead_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/a/aa/Bielefeld-CeBiTec_Biolector_lead_Balkendiagramm.jpeg" alt="Adjusting the detection limit"></a> | ||
| − | <figcaption>Fluorescence levels at three different stages of cultivation. Shown are levels after 60 minutes, 150 minutes and 650 minutes | + | <figcaption>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> The differences between inductions with various lead concentrations are really slight therefore this sensor needs further optimization which was not possible in this limited time. But as there is a fluorescence response to lead this sensor has the potential be work as expected. In the future a characterization in CFPS systems would be interesting. </p></br> | |
<h1>Mercury</h1> | <h1>Mercury</h1> | ||
Revision as of 14:51, 14 September 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.
Arsenic
in vivo
We tested an arsenic sensor with mRFP1 as reporter gene in vivo to confirm that the sensor is functional and test whether it is possible to detect the safety limit as defined by the WHO. We observed a reaction approximately five hours after addition of arsenic. The safety limit of 10 µg/L could clearly be distinguished from the negative control and the fluorescence signal increased up to a concentration of 500 µg/L. The signal in the presence of 1000 µg/L was slightly lower than in the presence of 500 µg/L.
in vitro
E. coli is resistant to arsenic because it posseses an efflux pump. The cell extract is not protected by such mechanisms, therefore we tested the effect of arsenic on the synthesis of sfGFP. We observed no significant effect for the relevant safety limits of 10 µg/L and 50 µg/L.
In order to test the arsenic sensor in our cell-free protein synthesis, we cloned a device that contains the arsenic operator between the T7 promoter and sfGFP with our optimized untranslated region (UTR). We tested this device in a cell extract that had been generated from cells expressing the arsenic repressor. We observed an induction when adding arsenic up to a concentration of 1.87 mg/L. As high arsenic concentrations inhibit the performance of the CFPS, we normalized the results for this effect. In the final application, this task is performed by our app.
Chromium
in vivo
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
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.
In vivo we could show that the adding different concentrations of copper has effects on the transcription levels of sfGFP.
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
In the following graphic the influences of different copper concentrations on the cell extact are shown
As shown above copper has no negatice influence on the functuality of our cell extact. Therefore a ralatively stable system for copper sensing is provided.
First tests with specific cell extract and different copper concentrations lead to further tests and normilisations.
Fluorescences normalised on coppers influence to the cell extract are shown above.
In addition to the native promoter operator device as measured above reporter constructs under the control of T7 promoter were tested.
Compared to the former fluorecence leves the T7 reporter device showed higer levels therefore a reporter device under the control of T7 promoter is more sutable for our CFPS.
after normalising on coppers influcence to the cell extract these differecnces were even more obvious
Lead
in vivo
In addition to these we constructed a sensor for lead detection. It consists of PbrR the repressor and the lead specific promoter PbrA. The promoter is regulated by the RcnR, which binds Pb-ions. As the former sensors this one encloses a sfGFP for detection via Fluorescence.
The differences between inductions with various lead concentrations are really slight therefore this sensor needs further optimization which was not possible in this limited time. But as there is a fluorescence response to lead this sensor has the potential be work as expected. In the future a characterization in CFPS systems would be interesting.
Mercury
in vivo
in vitro
Nickel
in vivo
