Composite

Part:BBa_K1758313

Designed by: Team Bielefeld-CeBiTec 2015   Group: iGEM15_Bielefeld-CeBiTec   (2015-08-30)

Chromium repressor under control of constitutive promoter and strong RBS,chromium responsive promote

Repressor for chromium induceble promoter crProm under the control of constitutive promoter (K608002)

Usage and Biology

Our sensor for chromium detection consists of chrB the repressor and the chromate specific promoter chrP. The promoter chrP is regulated by the repressor combined they are used for in vivo characterization , 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.

e used this repressor for our chromium sensor. Originaly its from Ochrobactrum triti ci5bvl1. We codon optimized it for use in E.coli. It is essential for our chromium sensor device BBa_K1758310. In combination with BBa_K1758312,BBa_K1758314 This part is used as our in vivo cerates our chromium sensor.

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 55
    Illegal NheI site found at 966
    Illegal NheI site found at 989
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 124
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

in vivo characterisation

Our sensors were cultivated in the BioLector. Due to the accuracy of this device we could measure our sample in duplicates.

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 characterisation

For the characterization of the chromium sensor with CFPS we used parts differing from that we used in vivo characterization. For the in vitro characterization we used a cell extract produced from cells which contain the plasmid (BBa_K1758310). The plasmid contains the gene chrB under the control of a constitutive promoter, so that the cell extract is enriched with repressor molecules. In addition to that we added plasmid-DNA of the chromium specific promoter chrP with 5’UTR-sfGFP under the control of T7-promoter (BBa_K1758314)to the cell extract. The T7-promoter is needed to get a better fluorescence expression.

repressor construct used for in vivo characterisation
To produce the cell extract for in vitro characterization a construct (BBa_K1758310 ) 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-chrP-UTR-sfGFP BBa_K1758314 used for in vitro characterization.
Adjusting the detection limit
Influence of different chromium concentrations on our crude cell extract. Error bars represent the standard deviation of three biological replicates.

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Categories
//function/sensor/metal
Parameters
None