Difference between revisions of "Part:BBa K3110051"
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<h1>Usage and Biology</h1>
 
<h1>Usage and Biology</h1>
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The regulatory element lldR binds to the lldRO1 and lldRO2 (the operator regions) and inhibit 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 instead lldR complex with L-Lactate remains bound to LldRO1 acting as a transcriptional activator. Interleukin-12. IL-12 is an anti-inflammatory cytokine, involved in the regulation of cell-mediated immune responses. Through a set of complex signaling cascade IL-12 activates our immune cells to reduce tumor growth. It is under the control of lactate sensitive O1PO2 to promote its production only when the cells encounter a thresold concentration of lactic acid. It is conjugated to YebF that mediates its secretion outside the cells into the cancer microenvironment. It also has a flag tag attached downstream to YebF so that its secretion outside the cells can be detected using anti-Flag antibodies.
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The regulatory element lldR binds to the lldRO1 and lldRO2 (the operator regions) and inhibit 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 instead lldR complex with L-Lactate remains bound to LldRO1 acting as a transcriptional activator. Interleukin-12. IL-12 is an anti-inflammatory cytokine, involved in the regulation of cell-mediated immune responses. Through a set of complex signaling cascade IL-12 activates our immune cells to reduce tumor growth. Its production is under the control of 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 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 anti-Flag antibodies.

Revision as of 15:09, 20 October 2019


lldRO1-J23117-lldRO2 Strong RBS YebF-IL12

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.

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 inhibit 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 instead lldR complex with L-Lactate remains bound to LldRO1 acting as a transcriptional activator. Interleukin-12. IL-12 is an anti-inflammatory cytokine, involved in the regulation of cell-mediated immune responses. Through a set of complex signaling cascade IL-12 activates our immune cells to reduce tumor growth. Its production is under the control of 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 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 anti-Flag antibodies.