Difference between revisions of "Part:BBa K2549025"
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This part is one of the downstream elements of our amplifier. Zinc finger 43.8 ([[Part:BBa_K2549048]]) 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.
 
This part is one of the downstream elements of our amplifier. Zinc finger 43.8 ([[Part:BBa_K2549048]]) 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.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2549025 SequenceAndFeatures</partinfo>
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<!-- Add more about the biology of this part here -->
 
<!-- Add more about the biology of this part here -->
 
===Biology===
 
===Biology===
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====previous reported engineered zinc finger-based transcription factors====
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=====It works as we designed =====
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Jason J. Lohmueller 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:aTF.jpeg|none|360px|thumb|Zinc finger-based transcription activator. Jason J. Lohmueller 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.'']]
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=====Previous reported engineered zinc finger-based transcription factors=====
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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:sTF.jpeg|none|360px|thumb|Zinc finger-based transcription repressor. Jason J. Lohmueller 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.'']]
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[[File:aTF.jpeg|none|360px|thumb|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.'']]  
  
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====artificial zinc fingers with modular DNA-binding domains====
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[[File:sTF.jpeg|none|360px|thumb|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.'']]
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JJ Collins et al have reported a synthetic biology framework based on orthogonal artificial zinc fingers<ref>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</ref>.
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[[File:ZF1.jpeg|none|540px|thumb|JJ Collins 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.'']]
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=====Artificial zinc fingers with modular DNA-binding domains=====
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Collins JJ et al have reported a synthetic biology framework based on orthogonal artificial zinc fingers<ref>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</ref>.
  
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[[File:ZF2.jpg|none|540px|thumb|JJ Collins 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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[[File:ZF1.jpeg|none|540px|thumb|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.'']]
  
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====ZF43.8-VP64 works as we designed====
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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.'']]
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2549025 SequenceAndFeatures</partinfo>
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2549025 parameters</partinfo>
 
<partinfo>BBa_K2549025 parameters</partinfo>
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===References===
 
===References===

Revision as of 14:24, 12 October 2018


ZF43.8-VP64

This part is one of the downstream elements of our amplifier. Zinc finger 43.8 (Part:BBa_K2549048) 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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 187
  • 1000
    COMPATIBLE WITH RFC[1000]


Biology

It works as we designed

@@@@

Previous reported engineered zinc finger-based transcription factors

Lohmueller JJ et al have reported a tunable zinc finger-based transcription framework in mammalian cells[1].

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[2].

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. ↑ 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
  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