Difference between revisions of "Team:Bielefeld-CeBiTec/Results/HeavyMetals"
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<p>Compared to the <i>in vivo</i> results, the response to arsenic was relatively small and we measured a high background signal. We assume that this is due to the different construct we used <i>in vitro</i>. This construct had been optimized for our CFPS by exchanging the natural promoter for the T7 promoter and exchanging mRFP1 for our optimized sfGFP. However, we assume that the repression in the presence of ArsR was not effective enough to observe a clear induction. The reason is most likely that the distance between the T7 promoter and the arsenic operator was too large. The distance was a result of our cloning strategy and would likely be suitable for <i>E. coli</i> promoters. However, the T7 promoter requires the operator to be very close for an efficient repression (<a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Results/HeavyMetals#Karig2012">Karig et al. 2012</a>). </p> | <p>Compared to the <i>in vivo</i> results, the response to arsenic was relatively small and we measured a high background signal. We assume that this is due to the different construct we used <i>in vitro</i>. This construct had been optimized for our CFPS by exchanging the natural promoter for the T7 promoter and exchanging mRFP1 for our optimized sfGFP. However, we assume that the repression in the presence of ArsR was not effective enough to observe a clear induction. The reason is most likely that the distance between the T7 promoter and the arsenic operator was too large. The distance was a result of our cloning strategy and would likely be suitable for <i>E. coli</i> promoters. However, the T7 promoter requires the operator to be very close for an efficient repression (<a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Results/HeavyMetals#Karig2012">Karig et al. 2012</a>). </p> | ||
| + | <p>In addition, we performed an experiment in which the arsenic repressor was not present in the reaction from the beginning, but was encoded on a second plasmid. The plasmid concentrations we used had been predicted by our model. In accordance with the aforementioned results, we observed no clear repression and addition of arsenic showed no effect. This experiment is discussed on the <a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Modeling/Application">Modeling pages</a></p> | ||
<h3>References</h3> | <h3>References</h3> | ||
Revision as of 16:53, 15 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
We choose to work with the chromosomal arsenic operon of E. coli, which was used by the team from Edinburgh in 2006. This operon encodes an efflux pump which confers resistance against arsenic. The expression is controlled by the repressor ArsR, which negatively autoregulates its own expression. AsIII can bind to three cysteine residues in ArsR. The resulting conformational change deactivates the repressor (Chen, Rosen 2014).
By placing a reporter gene downstream of arsR, an arsenic biosensor can be constructed. In this case, both the repressor and the reporter are under the control of the same promoter. In this respect, the arsenic sensor is different from the other heavy metal biosensors we worked with, as their repressors or activators are expressed constitutively. However, the genetic build-up of the arsenic sensor is well established. Consequently, we decided to keep this deviating design.
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.
Compared to the in vivo results, the response to arsenic was relatively small and we measured a high background signal. We assume that this is due to the different construct we used in vitro. This construct had been optimized for our CFPS by exchanging the natural promoter for the T7 promoter and exchanging mRFP1 for our optimized sfGFP. However, we assume that the repression in the presence of ArsR was not effective enough to observe a clear induction. The reason is most likely that the distance between the T7 promoter and the arsenic operator was too large. The distance was a result of our cloning strategy and would likely be suitable for E. coli promoters. However, the T7 promoter requires the operator to be very close for an efficient repression (Karig et al. 2012).
In addition, we performed an experiment in which the arsenic repressor was not present in the reaction from the beginning, but was encoded on a second plasmid. The plasmid concentrations we used had been predicted by our model. In accordance with the aforementioned results, we observed no clear repression and addition of arsenic showed no effect. This experiment is discussed on the Modeling pages
References
Chen, Jian; Rosen, Barry P. (2014): Biosensors for inorganic and organic arsenicals. In Biosensors 4 (4), pp. 494–512. DOI: 10.3390/bios4040494.
Karig, David K.; Iyer, Sukanya; Simpson, Michael L.; Doktycz, Mitchel J. (2012): Expression optimization and synthetic gene networks in cell-free systems. In Nucleic acids research 40 (8), pp. 3763–3774. DOI: 10.1093/nar/gkr1191.
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 higher levels therefore a reporter device under the control of T7 promoter is more suitable 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 work as expected. In the future a characterization in CFPS systems would be interesting.
Mercury
in vivo
One of the already existing sensors we use for our system is the mercury sensor consisting of MerR the activator and the mercury specific promoter MerT. The promoter is regulated by the MerR, which binds Hg-ions. Similar to the former sensors we added a sfGFP for detection via fluorescence.
In vivo data show a highly significant, well working sensor which even reacts to concentrations which are mentioned as drinking water guidelines by the WHO.
The mercury detection was measured during the cultivation of E. coli KRX at 37 °C. The strain contains the plasmid with the activator MerRunder the control of a constitutive promoter and the specific promoter with operator site which reacts to the activator with bound Hg-ions. The specific promoter is in front of sfGFP for measurment , so the mercury in the medium is detected directly.In vivo this sensor devise shows a fast answer to occurrence of his heavy metal contrary to the other sensor systems in vivo.
Therefore we tested our sensor in vitro to check if an already functioning highly optimized sensor provides required data for guideline detections
in vitro
Nickel
in vivo
In addition to these we aimed to construct a sensor for nickel detection. It consists of RcnR the repressor and the nickel specific promoter RcnA. The promoter is regulated by the RcnR, which binds Ni-ions. As the former sensors this one encloses a sfGFP for detection via Fluorescence.
With this sensor no production of sfGFp via fluorescence level change could be detected. Therefore this sensor is not suitable for approach. Therefore no in vitro data using CFPS were taken.