Generator

Part:BBa_K1758353

Designed by: Team Bielefeld-CeBiTec 2015   Group: iGEM15_Bielefeld-CeBiTec   (2015-09-08)

Nickel repressor under control of constitutive promoter and strong RBS and Nickel responsive promote

Nickel repressor under the control of a constitutive promoter with the induceble promoter, a 5´untranslated region in front of the sfGFP which is used for detection.

Usage and Biology

Our Nickel biosensor consists of parts of the rcn-operon from E. coli which codes for a nickel- and cobalt-efflux system. This system is highly sensitive to nickel. In absence of nickel or cobalt RcnR binds to the operator and inhibits the nickel responsive promoter. With Ni(II)-ions present the repression of the promoter RcnA will be reversed, because the repressor RcnR binds nickel-ions and cannot attach to the DNA (EPA et al., 2013; Blaha et al., 2011; Iwig et al., 2006). For our biosensor we construct the part ( BBa_K1758353 by using the basic construction showed in . For this part we used the repressor RcnR under control of a constitutive promoter ( BBa_K1758350 ) and the nickel specific promoter RcnA with a 5’UTR in front of sfGFP ( BBa_K1758352 ) as reporter protein.

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

in vivo

We aimed to construct a sensor for nickel detection. It consists of rcnR the repressor and the nickel specific promoter prcnA. The promoter is regulated by the RcnR, which binds Ni2+-ions. As the former sensors this one encloses a sfGFP for detection via fluorescence.

Our nickel biosensor consists of parts of the rcn-operon from E. coli, which encodes a nickel- and cobalt-efflux system. This system is highly sensitive to nickel. In absence of nickel or cobalt RcnR binds to the operator and inhibits the nickel responsive promoter. With Ni2+-ions present the repression of the promoter prcnA will be reversed, because the repressor RcnR binds Ni2+-ions and cannot attach to the DNA. For our biosensor we construct the part BBa_K1758353 by using the basic construction shown in figure 1. For this part we used the repressor RcnR under control of a constitutive promoter ( BBa_K1758350 ) and the nickel specific promoter PrcnA with a 5’UTR in front of sfGFP ( BBa_K1758352 ) as reporter protein.

Figure 1: The concept of our in vivo nickel sensor ( BBa_K1758354), which consists of the activator under the control of a constitutive promoter ( BBa_K1758350)and the operator and promoter sequence of the nickel inducible promoter. An untranslated region in front of the sfGFP, which is used for detection, enhances its expression ( BBa_K1758352)
Adjusting the detection limit
Figure 2: Time course of the induction of a nickel biosensor with sfGFP for different nickel concentrations in vivo. The data are measured with BioLector and normalized on OD600. Error bars represent the standard deviation of two biological replicates.
Figure 3: 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.

We tested our nickel sensor with sfGFP as reporter gene, to test the functionality of the system. Moreover we tested different concentrations. The kinetic of our sensors response to different nickel concentrations is shown in figure 2. The first five hours show a strong decrease in fluorescence. After that there is a slight increase in fluorescence. Starting levels of fluorescence are not reached. For better visualization the kinetics of figure 2 are represented as bars in figure 3. A fluorescence level difference for 60 min, 150 min and 650 min is represented.

The data for our nickel sensor show a trend that differs for that of the other sensors. There is no indication for a working sensor in vivo (Figure 2 and 3). There is a fluorescence signal, but it decreases in the first five hours. After reaching a minimum the fluorescence increases slowly. Additionally, there is no difference in fluorescence as response to various nickel concentration. Nickel could influence the cells and thereby caused a precipitation, which could result in decrease of fluorescence. With this sensor no production of sfGFP via fluorescence level change could be detected. Therefore, this sensor is not suitable for our approach. Due to this observation, no in vitro data using CFPS were taken.


To summarize

With this sensor no production of sfGFP via fluorescence level change could be detected. Therefore, this sensor is not suitable for our approach. Consequently, no in vitro tests were performed. To create a working sensor based on this concept further optimization is needed.

Refrences

Blaha, Didier; Arous, Safia; Blériot, Camille; Dorel, Corinne; Mandrand-Berthelot, Marie-Andrée; Rodrigue, Agnès (2011): The Escherichia coli metallo-regulator RcnR represses rcnA and rcnR transcription through binding on a shared operator site: Insights into regulatory specificity towards nickel and cobalt. In Biochimie 93 (3), pp. 434–439. DOI: 10.1016/j.biochi.2010.10.016.

EPA, U. S.; OAR; Office of Air Quality Planning and Standards (2013): Nickle Compounds | Technology Transfer Network Air Toxics Web site | US EPA. Available online at http://www.epa.gov/airtoxics/hlthef/nickel.html, updated on 10/18/2013, checked on 9/10/2015.

Iwig, Jeffrey S.; Rowe, Jessica L.; Chivers, Peter T. (2006): Nickel homeostasis in Escherichia coli - the rcnR-rcnA efflux pathway and its linkage to NikR function. In Molecular microbiology 62 (1), pp. 252–262. DOI: 10.1111/j.1365-2958.2006.05369.x.

[edit]
Categories
//function/sensor/metal
Parameters
None