Composite

Part:BBa_K1758344

Designed by: Team Bielefeld-CeBiTec 2015   Group: iGEM15_Bielefeld-CeBiTec   (2015-08-29)

Mercury responsive promoter with T7-promoter and UTR-sfGFP

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

Usage and Biology

The promoter PmerT is regulated by the MerR, which binds Hg2+-ions. Similar to the former sensors we added a sfGFP for detection via fluorescence. It is based on BBa_K346001 desinged by team Peking 2010.For the in vitro characterization we used a cell extract out of cells which contain the Plasmid ( BBa_K1758340). In addition to that we added Plasmid-DNA of the mercury specific promoter merT with 5’UTR-sfGFP under the control of T7-promoter ( BBa_K1758344)to the cell extract. The T7-promoter is needed to get a better fluorescence expression.

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


in vitro

For the characterization of the mercury sensor with CFPS we used parts differing from that we used in the in vivo characterization. For the in vitro characterization we used a cell extract out of cells, which contained the plasmid ( BBa_K1758340)(figure 5). In addition, we added plasmid DNA to the cell extract. This plasmid consisted of the mercury specific promoter pmerT with 5’-UTR-sfGFP. The entire sequence was placed under the control of of T7-promoter ( BBa_K1758344)(figure 6). The T7-promoter is needed to get a better fluorescence expression.

repressor construct used for in vitro characterisation
Figure 5: To produce the cell extract for in vitro characterization a construct (BBa_K175840) 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-PmerT-UTR-sfGFP BBa_K175844 used forin vitro characterization.
Figure 7: Influence of different mercury concentrations on our crude cell extract. Error bars represent the standard deviation of three biological replicates.
Figure 8: Mercury specific cell extract made from E. coli cells, which have already expressed the activator before cell extract production. Induction of mercury inducible promoter without T7 in front of the operator site with different mercury concentrations. Error bars represent the standard deviation of three biological replicates.
Figure 9: Mercury specific cell extract made from E. coli cells, which have already expressed the activator before cell extract production. Induction of mercury inducible promoter without T7 in front of the operator site with different mercury concentrations. Error bars represent the standard deviation of three biological replicates.

In vitro this sensor showed good results. The fluorescence level was high at low concentrations. Additionally, it showed that the expression level at 6 µg/L (Guideline of WHO for Mercury) reached the maximal signal. This result indicated the potential for measurement of concentrations under 6 µg/L.To confirm this hypothesis, it takes more experiments and tests with lower concentrations. Due to the high expression of sfGFP at low concentrations and the same expression level at different concentrations, it is not possible to quantify mercury with CFPS analyses . , Our model predicted this observation. During the measurement we noticed that the heavy metals have negative influences on the cell extract. Because of this fact, we used a correction factor, which resulted from the heavy metals influence on the CFPS system. This already optimized sensor showed the high potential of optimized sensors in CFPS.

References

iGEM Team Peking 2010

Brown, Nigel L.; Stoyanov, , Jivko V.;Kidd,Stephen P.;Hobman; Jon L. (2003): The MerR family of transcriptional regulators. In FEMS Microbiology Reviews, 27 ( 2) pp.145-163.

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Categories
//function/sensor/metal
Parameters
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