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> | + | <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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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.
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.