Difference between revisions of "Part:BBa K3110053"

 
 
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lldRO1 and lldRO2 are the operator regions which bind to the regulator element lldR and inhibit transcription. Upon binding of L-Lactate to lldR, however, this transcriptional suppression is lost and instead lldR complex with L-Lactate remains bound to lldRO1 acting as a transcriptional activator. By using RBS of different strengths we are trying to find out the optimum construct which will give output (in this case the output is YebF-IL12) that can efficiently discriminate cancer vs non-cancer micro-environments and will lead to secretion of IL12 (natural immuno-modulator) and eventually reduction of tumour size.
 
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===Usage and Biology===
 
  
 
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<h1>Usage and Biology</h1>
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The regulatory element lldR binds to the lldRO1 and lldRO2 (the operator regions) and inhibits transcription. J23117 is a promoter intercalated between the operators. lldR dimer represses the transcription, possibly by forming a DNA loop which doesn’t allow the RNA polymerase to bind the promoter. Upon binding of L-Lactate to lldR, however, this transcriptional suppression is lost and the lldR complex with L-Lactate remains bound to LldRO1, acting as a transcriptional activator. IL12 is an anti-inflammatory cytokine involved in the regulation of cell-mediated immune responses. Through a complex signaling cascade, IL12 activates our immune cells to reduce tumor growth. Its production is under the control of the lactic acid promoter, lldPRDp, which has been optimised to respond specifically to lactic acid concentrations in the milieu of colon cancer cells. YebF is a secretory protein present in E. coli. We have attached the IL12 sequence in the C-terminus of YebF so that IL12 also gets secreted out of E. coli along with YebF. A flag tag is attached downstream to YebF so that its secretion outside the cells can be detected using Flag antibodies.
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<h1>Characterization</h1>
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Due to time constraint and consecutive failures, we couldn’t make this construct and characterize it.

Latest revision as of 11:35, 21 October 2019


lldRO1-J23117-lldRO2-Weak RBS-YebF IL12

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 78
    Illegal NheI site found at 101
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

The regulatory element lldR binds to the lldRO1 and lldRO2 (the operator regions) and inhibits transcription. J23117 is a promoter intercalated between the operators. lldR dimer represses the transcription, possibly by forming a DNA loop which doesn’t allow the RNA polymerase to bind the promoter. Upon binding of L-Lactate to lldR, however, this transcriptional suppression is lost and the lldR complex with L-Lactate remains bound to LldRO1, acting as a transcriptional activator. IL12 is an anti-inflammatory cytokine involved in the regulation of cell-mediated immune responses. Through a complex signaling cascade, IL12 activates our immune cells to reduce tumor growth. Its production is under the control of the lactic acid promoter, lldPRDp, which has been optimised to respond specifically to lactic acid concentrations in the milieu of colon cancer cells. YebF is a secretory protein present in E. coli. We have attached the IL12 sequence in the C-terminus of YebF so that IL12 also gets secreted out of E. coli along with YebF. A flag tag is attached downstream to YebF so that its secretion outside the cells can be detected using Flag antibodies.

Characterization

Due to time constraint and consecutive failures, we couldn’t make this construct and characterize it.