Difference between revisions of "Part:BBa K1758324"
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<p>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 <i>in vitro</i> transcription translation approach.</p>
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<p>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 <a href="http://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals" ><i>in vitro</i> transcription translation approach.</a> </p>

Revision as of 21:48, 16 September 2015

Usage and Biology

For our copper sensor we used the native operator of cooper homeostasis from E.coli K12. This includes the promoter (CopAP) and its regulator CueR. CueR is a MerR like regulator, which stimulates the transcription of CopA, a P-type ATPase pump (Outten et al. 2000). CopA is the central component in obtaining copper homeostasis, it exports free copper from cytoplasm to the periplasm. This is enable by Copper induced activation of the operon transcription via CueR. The CueR-Cu+ is the DNA-binding transcriptional dual regulator which activates transcription(Yamamoto, Ishihama 2005) To sum it up CueR regulon plays an important role in aerobic copper tolerance in E.coli(Grass, Rensing 2001).In BBa_K1758324 we combined the codon optimized CueR (BBa_K1758320) under the control of a constitutive promoter with strong RBS(BBa_K608002)with CopAP and sfGFP (BBa_K1758321) for measuring output signals. Through the addition of a 5’UTR before the sfGFP we optimized the expression of sfGFP and increased.

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 475
    Illegal SapI.rc site found at 625


Results

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 copper biosensor with sfGFP for different copper concentrations in vivo. The data are measured with BioLector and normalized on OD600. Error bars represent the standard deviation of two biological replicates.

In vivo we could show that the adding different concentrations of copper has effects on the transcription levels of sfGFP.

Adjusting the detection limit
Fluorescence levels at three different stages of cultivation. Shown are levels after 60 minutes, 150 minutes and 650 minutes.

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.