Difference between revisions of "Part:BBa K2486014"
 
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https://static.igem.org/mediawiki/2017/b/bd/T--USP-Brazil--Detecttion_Iron_Lactate_concept_%287%29.PNG
 
https://static.igem.org/mediawiki/2017/b/bd/T--USP-Brazil--Detecttion_Iron_Lactate_concept_%287%29.PNG
  
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The data of the validation of this part in the presence of DtxR and Fe2+ is described in part
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The data of the validation of this part in the presence of DtxR and Fe2+ is described in part [https://parts.igem.org/wiki/index.php?title=Part:BBa_K2486021 BBa_K2486021] and [https://parts.igem.org/Part:BBa_K2486014 here].
  
  

Latest revision as of 03:00, 2 November 2017


PdtxR reporter circuit (GFP)

This module works as a reporter for lactate an tetracycline presence. The circuit's key part is the PdtxR promoter (BBa_K2486175),a synthetic promoter based on BBa_J23108 promoter from Anderson Promoter Collection with a DtxR binding site carefully added to it. We adopted dtxR operator in our promoter instead of fur operator because of dtxR different operator sequence, wich reduces its interaction with endogenous sites for this regulator. While the fur Fur regulator is derived form E. coli the DtxR is derived from Corynebacterium diphtheriae

  1. BBa_B0034: a strong RBS from [the https://parts.igem.org/Ribosome_Binding_Sites/Prokaryotic/Constitutive/Community_Collection Community Collection]
  2. BBa_K2486024: a 6 base pair spacer for optimal expression.
  3. BBa_K082003: a GFP with LVA tag

Characterization

T--USP-Brazil--Detecttion_Iron_Lactate_concept_%287%29.PNG

The data of the validation of this part in the presence of DtxR and Fe2+ is described in part BBa_K2486021 and here.


PdtxR promoter activity in the presence of DtxR regulator

T--USP-Brazil--Detecttion_Iron_Lactate_dtxR%281%29.png

Promoter activity (GFP/OD600) under different concentrations of Iron Chloride (Fe2+) shows a decrease in promoter activity when (Fe2+) is added to the system, wich is expected by the funciotn mechanism of the regulator. The binding of the DtxR protein to iron enhances it's affinity to the DNA repressing the GFP expression.

T--USP-Brazil--Detecttion_Iron_Lactate_dtxR%282%29.png

After 24h the results are similar to the kinectic analysis of expression.

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 716