Difference between revisions of "Part:BBa K2549026"

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===Characterization===
 
===Characterization===
 
=====It works as we designed.=====
 
=====It works as we designed.=====
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[[File:aTF-test.png|none|480px|thumb|'''Fig.1 Interaction between transcriptional activators and their binding sites. A degradable EGFP (d2EGFP) is linked downstream the promoter to indicate the expression level of it. DBD, DNA binding domain which is zinc finger in our assay. AD or SD, activating- or silencing-form transcriptional domain. RE, responsive elements. RFI, relative fluorescence intensity.''']]
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[[File:aTF-test.png|none|480px|thumb|'''Fig.1 Interaction between transcriptional activators and their binding sites. A degradable EGFP (d2EGFP) is linked downstream the promoter to indicate the expression level of it. DBD, DNA binding domain which is zinc finger in our assay. AD or SD, activating- or silencing-form transcriptional domain. RE, responsive elements. MFI, median fluorescence intensity.''']]
  
 
Flow cytometry results suggest that the transcriptional activators can effectively activate the responsive promoters with high specificity and high orthogonality.
 
Flow cytometry results suggest that the transcriptional activators can effectively activate the responsive promoters with high specificity and high orthogonality.

Revision as of 13:29, 17 October 2018


8*ZF21.16-minCMV

This part is one of the response elements of our amplifier, also executing the combiner function. 8*ZF21.16 binding sites (Part:BBa_K2446015) is assembled using two 4*ZF21.16 binding sites (Part:BBa_K2446008) with a biobrick scar between them. Minimal CMV (Part:BBa_K2549049) is a promotor providing very low basal expression and high maximal expression after induction. This part can switch on the expression of gene downstream after induced by our zinc finger-based transcription activator.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Biology

Synthetic promotor operators regulated by artificial zinc finger-based transcription factors

Khalil AS et al have reported several synthetic promotor operators which can interact with artificial zinc finger-based transcription factors with high specificity and high orthogonality[1].

Khalil AS et al stated:sTFs constructed from OPEN-engineered ZFs are orthogonal to one another. sTF43-8 activated noncognate Promoter21-16 due to the fortuitous creation of a sequence that is significantly similar to the binding sequence of 43-8, when the downstream BamHI restriction site is considered.

Characterization

It works as we designed.
Fig.1 Interaction between transcriptional activators and their binding sites. A degradable EGFP (d2EGFP) is linked downstream the promoter to indicate the expression level of it. DBD, DNA binding domain which is zinc finger in our assay. AD or SD, activating- or silencing-form transcriptional domain. RE, responsive elements. MFI, median fluorescence intensity.

Flow cytometry results suggest that the transcriptional activators can effectively activate the responsive promoters with high specificity and high orthogonality.

Fig.2 OR gate based on 8*ZF21.16-minCMV. A degradable EGFP (d2EGFP) is linked downstream the promoter to indicate the expression level of it. DBD, DNA binding domain which is zinc finger in our assay. AD or SD, activating- or silencing-form transcriptional domain. RE, responsive elements. RFI, relative fluorescence intensity.

Flow cytometry results indicate that when at least one of the transcriptional activators exist the expression of the promoter can be activated.


References

  1. ↑ A synthetic biology framework for programming eukaryotic transcription functions. Khalil AS, Lu TK, Bashor CJ, ..., Joung JK, Collins JJ. Cell, 2012 Aug;150(3):647-58 PMID: 22863014; DOI: 10.1016/j.cell.2012.05.045