Difference between revisions of "Part:BBa M50499:Design"
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<partinfo>BBa_M50499 short</partinfo>
 
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<partinfo>BBa_M50499 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_M50499 SequenceAndFeatures</partinfo>
  
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For our experiments, we placed this part upstream from GFP in order to assay expression levels through fluorescent intensity. This construct is an adaption of the natural lac operon, which is positively regulated by CAP, since the CAP binding site is naturally upstream of the RNA polymerase binding site. In this construct, we put the CAP binding site downstream in order to repress transcription when CAP binds due to steric hindrance.
  
 
===Design Notes===
 
===Design Notes===

Revision as of 23:50, 11 December 2018


Catabolite Activator Protein (CAP) Repressed Promoter



Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1059
    Illegal PstI site found at 688
    Illegal PstI site found at 849
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 144
    Illegal NheI site found at 167
    Illegal PstI site found at 688
    Illegal PstI site found at 849
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1059
    Illegal PstI site found at 688
    Illegal PstI site found at 849
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1059
    Illegal PstI site found at 688
    Illegal PstI site found at 849
  • 1000
    COMPATIBLE WITH RFC[1000]


For our experiments, we placed this part upstream from GFP in order to assay expression levels through fluorescent intensity. This construct is an adaption of the natural lac operon, which is positively regulated by CAP, since the CAP binding site is naturally upstream of the RNA polymerase binding site. In this construct, we put the CAP binding site downstream in order to repress transcription when CAP binds due to steric hindrance.

Design Notes

We were unsure whether to put the CRP binding site immediately downstream or with a buffer sequence. We ended up putting it right after the promoter.


Source

IGEM part: BBa_S05450 = constitutive promoter IGEM part: BBa_M36547 = CRP binding site

Paper: www.ncbi.nlm.nih.gov/pmc/articles/PMC338411/

References