Difference between revisions of "Part:BBa K2549030"

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===Biology===
 
===Biology===
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=====Our characterization=====
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[[File:sTF-test.png|none|360px|thumb|'''Interaction between transcriptional repressors and their binding sites.''' A degradable EGFP (d2EGFP) is produced downstream the promoter to indicate the output strength. Experiments were conducted and analyzed as previous reported<ref>http://2017.igem.org/Team:Fudan/Demonstrate</ref>. DBD, DNA binding domain which is zinc finger in our assay. SD, silencing-form transcriptional domain; we used KRAB for the experiments in this figure. RE, responsive elements. MFI, median fluorescence intensity.]]
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Flow cytometry results suggest that the transcriptional repressors can effectively inhibit the promoters with responsive elements. Please visit http://2018.igem.org/Team:Fudan/Demonstrate for a brief introduction of our project.
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=====Synthetic promotor operators regulated by artificial zinc finger-based transcription factors=====
 
=====Synthetic promotor operators regulated by artificial zinc finger-based transcription factors=====
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[[File:ZF2.jpg|none|240px|thumb|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.'']]
 
[[File:ZF2.jpg|none|240px|thumb|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.'']]
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===Characterization===
 
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=====It works as we designed =====
 
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[[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.''']]
 
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It is obvious that the three zinc finger-based operators can be significantly repressed by their specific zinc finger-based transcriptional repressor, respectively.
 
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Latest revision as of 18:46, 6 November 2018

8*ZF42.10-CMV

This part is one of the response elements of our amplifier, also executing the combiner function. 8*ZF42.10 binding sites (Part:BBa_K2549012) is assembled using two 4*ZF42.10 binding sites (Part:BBa_K2446005) 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.

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

Our characterization
Interaction between transcriptional repressors and their binding sites. A degradable EGFP (d2EGFP) is produced downstream the promoter to indicate the output strength. Experiments were conducted and analyzed as previous reported[1]. DBD, DNA binding domain which is zinc finger in our assay. SD, silencing-form transcriptional domain; we used KRAB for the experiments in this figure. RE, responsive elements. MFI, median fluorescence intensity.

Flow cytometry results suggest that the transcriptional repressors can effectively inhibit the promoters with responsive elements. Please visit http://2018.igem.org/Team:Fudan/Demonstrate for a brief introduction of our project.


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[2].

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.


References

  1. ↑ http://2017.igem.org/Team:Fudan/Demonstrate
  2. ↑ 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