Difference between revisions of "Part:BBa K2549029"

(→‎Characterization)
(→‎Characterization)
Line 22: Line 22:
 
=====It works as we designed =====
 
=====It works as we designed =====
  
−
[[File:sTF-test.png|none|480px|thumb|'''Flow cytometry results of transcriptional repressor interaction with zinc finger-based binding sites. DBD, DNA binding domain which is zinc finger in our assay. SD, silencing-form transcriptional domain. RE, responsive elements. MFI, median fluorescence intensity.''']]
+
[[File:sTF-test.png|none|480px|thumb|'''Fig.1 Flow cytometry results of transcriptional repressor interaction with zinc finger-based binding sites. DBD, DNA binding domain which is zinc finger in our assay. SD, silencing-form transcriptional domain. RE, responsive elements. MFI, median fluorescence intensity.''']]
  
 
It is obvious that the three zinc finger-based operators can be significantly repressed by their specific zinc finger-based transcriptional repressor, respectively.
 
It is obvious that the three zinc finger-based operators can be significantly repressed by their specific zinc finger-based transcriptional repressor, respectively.
 +
 +
[[File:NORgate.png|none|480px|thumb|'''Fig.2 ZF21.16-CMV-based NOR gate. 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. SD, silencing-form transcriptional domain. RE, responsive elements. RFI, relative fluorescence intensity.''']]
 +
 +
Flow cytometry results indicate that only when neither of the transcriptional repressor exists can the d2EGFP be expressed.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 13:42, 17 October 2018

8*ZF21.16-CMV

This part is one of the response elements of our amplifier, also executing the combiner function. 8*ZF21.16 binding sites is assembled using two 4*ZF21.16 binding sites (Part:BBa_K2446008) with a biobrick scar between them. CMV (Part:BBa_K2549050) is a promotor which has a high-level constitutive expression. This part can switch off the expression of gene downstream after induced by our zinc finger-based transcription repressor.

This part is also served as an improvement version of Part:BBa_K2446030 of iGEM-team [http://2017.igem.org/Team:Fudan/Composite_Part Fudan 2017]. We placed responsive elements of zinc finger-based transcription factors on the 5' end of the promotor to lower the impact they may have on the basal expression of the promotor. Besides, CMV promotor performs a higher-level expression than SV40 promotor. For more details, please refer to our [http://2018.igem.org/Team:Fudan/Improve Parts improvement page].

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 Flow cytometry results of transcriptional repressor interaction with zinc finger-based binding sites. DBD, DNA binding domain which is zinc finger in our assay. SD, silencing-form transcriptional domain. RE, responsive elements. MFI, median fluorescence intensity.

It is obvious that the three zinc finger-based operators can be significantly repressed by their specific zinc finger-based transcriptional repressor, respectively.

Fig.2 ZF21.16-CMV-based NOR gate. 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. SD, silencing-form transcriptional domain. RE, responsive elements. RFI, relative fluorescence intensity.

Flow cytometry results indicate that only when neither of the transcriptional repressor exists can the d2EGFP be expressed.


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