Difference between revisions of "Part:BBa K2549023"

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===Biology===
 
===Biology===
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=====Our characterization=====
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[[File:Synnotch-GV2.png|right|300px|thumb|'''Flow cytometry results of an intercellular OR gate.''' RFI: output from the Combiner after activation was divided by the value before activation. Similar to tTAA, transcriptional activator GV2 works well. The TRE3GV or URE2G promoters in the Amplifer receive signals from intercellular tTAA or GV2, respectively. Both Amplifiers would produce ZF21.16-VP64, which could transduce signal to the Combiner which has ZF21.16 responsive elements (the version of 8 copies of responsive elements is the best). More details please visit http://2018.igem.org/Team:Fudan/Results and http://2018.igem.org/Team:Fudan/Measurement .]]
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[[File:aTF-test.png|none|420px|thumb|'''Interaction between transcriptional activators 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. AD, activating-form transcriptional domain; we used VP64 for the experiments in this figure. RE, responsive elements. MFI, median fluorescence intensity.]]
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Flow cytometry results suggest that the transcriptional activators can specifically activate the promoters with responsive elements, orthogonally. Please visit http://2018.igem.org/Team:Fudan/Demonstration for a brief introduction of our project.
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=====Previous reported engineered zinc finger-based transcription factors=====
 
=====Previous reported engineered zinc finger-based transcription factors=====
 
Lohmueller JJ et al have reported a tunable zinc finger-based transcription framework in mammalian cells<ref>A tunable zinc finger-based framework for Boolean logic computation in mammalian cells. Lohmueller JJ, Armel TZ, Silver PA. Nucleic Acids Res, 2012 Jun;40(11):5180-7  PMID: 22323524; DOI: 10.1093/nar/gks142</ref>.
 
Lohmueller JJ et al have reported a tunable zinc finger-based transcription framework in mammalian cells<ref>A tunable zinc finger-based framework for Boolean logic computation in mammalian cells. Lohmueller JJ, Armel TZ, Silver PA. Nucleic Acids Res, 2012 Jun;40(11):5180-7  PMID: 22323524; DOI: 10.1093/nar/gks142</ref>.
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[[File:ZF2.jpg|none|240px|thumb|Collins JJ 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|Collins JJ 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: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.''']]
 
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Flow cytometry results suggest that the transcriptional activators can effectively activate the responsive promoters with high specificity and high orthogonality.
 
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[[File:ORgate.png|none|480px|thumb|'''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.''']]
 
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Flow cytometry results indicate that when at least one of the transcriptional activators exist the expression of the promoter can be activated.
 
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 19:22, 17 October 2018


ZF21.16-VP64

This part is one of the downstream elements of our amplifier. Zinc finger 21.16 (Part:BBa_K2549046) is a synthetic engineered DNA binding domain with high sequence specificity and high orthogonality with other zinc finger proteins. VP64 (Part:BBa_K2549057) is a tetrameric VP16 transcription activator which shows ultrahigh transcription activation function. A SV40NLS domain (Part:BBa_K2549054) is placed on the N terminal of VP64 to guide the fusion protein to enter in the nucleus. A G4S linker (Part:BBa_K2549053) is set between ZF21.16 and SV40NLS. This part can also be utilized by other iGEM teams as zinc finger-based transcription activators to construct their own genetic circuits.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 304
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Biology

Our characterization
Flow cytometry results of an intercellular OR gate. RFI: output from the Combiner after activation was divided by the value before activation. Similar to tTAA, transcriptional activator GV2 works well. The TRE3GV or URE2G promoters in the Amplifer receive signals from intercellular tTAA or GV2, respectively. Both Amplifiers would produce ZF21.16-VP64, which could transduce signal to the Combiner which has ZF21.16 responsive elements (the version of 8 copies of responsive elements is the best). More details please visit http://2018.igem.org/Team:Fudan/Results and http://2018.igem.org/Team:Fudan/Measurement .
Interaction between transcriptional activators 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. AD, activating-form transcriptional domain; we used VP64 for the experiments in this figure. RE, responsive elements. MFI, median fluorescence intensity.

Flow cytometry results suggest that the transcriptional activators can specifically activate the promoters with responsive elements, orthogonally. Please visit http://2018.igem.org/Team:Fudan/Demonstration for a brief introduction of our project.


Previous reported engineered zinc finger-based transcription factors

Lohmueller JJ et al have reported a tunable zinc finger-based transcription framework in mammalian cells[2].

Zinc finger-based transcription activator. Lohmueller JJ et al stated: We generate ZF-TF activators and repressors and demonstrate a novel, general method to tune ZF-TF response by fusing ZF-TFs to leucine zipper homodimerization domains. We describe 15 transcriptional activators that display 2- to 463-fold induction and 15 transcriptional repressors that show 1.3- to 16-fold repression.
Zinc finger-based transcription repressor. Lohmueller JJ et al stated: We generate ZF-TF activators and repressors and demonstrate a novel, general method to tune ZF-TF response by fusing ZF-TFs to leucine zipper homodimerization domains. We describe 15 transcriptional activators that display 2- to 463-fold induction and 15 transcriptional repressors that show 1.3- to 16-fold repression.
Artificial zinc fingers with modular DNA-binding domains

Collins JJ et al have reported a synthetic biology framework based on orthogonal artificial zinc fingers[3].

Collins JJ et al stated: Eukaryotic transcription factors (TFs) perform a variety of molecular functions to control promoters and facilitate the operation of genetic networks (top panel). Zinc fingers (ZFs) are modular domains found in many eukaryotic TFs that make sequence-specific contacts with DNA. Artificial ZF arrays were used as core building blocks for constructing synthetic TFs (sTFs) and gene circuitry in S. cerevisiae (bottom panel). The use of artificial ZF domains permits a fully decomposed design of a sTF, for which the molecular component properties are accessible, modular, and tunable (red italicized). The independent control of these component properties enables the systematic construction and modulation of transcriptional behavior. AD, transcriptional activation domain; GOI, gene of interest; REs, regulatory elements.
Collins JJ 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 tunable zinc finger-based framework for Boolean logic computation in mammalian cells. Lohmueller JJ, Armel TZ, Silver PA. Nucleic Acids Res, 2012 Jun;40(11):5180-7 PMID: 22323524; DOI: 10.1093/nar/gks142
  3. ↑ 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