Difference between revisions of "Part:BBa K2549038"
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This part is one of the downstream elements of our amplifier. It is constructed by fusing CfaC ([[Part:BBa_K2549010]]), ZF21.16C ([[Part:BBa_K2549012]]) and NLS ([[Part:BBa_K2549054]]), from N terminal to C terminal. CfaC is the C-terminal fragment of Cfa which is a consensus sequence from an alignment of 73 naturally occurring DnaE inteins that are predicted to have fast splicing rates. ZF21.16C is the C-terminal fragment of the zinc finger whose recognition helices for three-finger arrays are substituted by the reported synthetic zinc finger 21.16 residues on the basis of the BCR_ABL-1 artificial zinc finger<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>. NLS is a short nuclear location sequence from SV40 large T antigen. When coexpressed with VP64-ZF21.16N-CfaN ([[Part:BBa_K2549036]]) in the same cell, both fusions are formed and a transcription activating function is executed. Also, when coexpressed with KRAB-ZF21.16N-CfaN ([[Part:BBa_K2549037]]) in the same cell, both fusions are formed and a transcription repressing function is executed.
 
This part is one of the downstream elements of our amplifier. It is constructed by fusing CfaC ([[Part:BBa_K2549010]]), ZF21.16C ([[Part:BBa_K2549012]]) and NLS ([[Part:BBa_K2549054]]), from N terminal to C terminal. CfaC is the C-terminal fragment of Cfa which is a consensus sequence from an alignment of 73 naturally occurring DnaE inteins that are predicted to have fast splicing rates. ZF21.16C is the C-terminal fragment of the zinc finger whose recognition helices for three-finger arrays are substituted by the reported synthetic zinc finger 21.16 residues on the basis of the BCR_ABL-1 artificial zinc finger<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>. NLS is a short nuclear location sequence from SV40 large T antigen. When coexpressed with VP64-ZF21.16N-CfaN ([[Part:BBa_K2549036]]) in the same cell, both fusions are formed and a transcription activating function is executed. Also, when coexpressed with KRAB-ZF21.16N-CfaN ([[Part:BBa_K2549037]]) in the same cell, both fusions are formed and a transcription repressing function is executed.
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<span class='h3bb'>Sequence and Features</span>
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===Biology===
 
===Biology===
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====Boolean logic gates via split zinc finger-based transcription factors====
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=====It works as we designed =====
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coexpressed with VP64-ZF21.16N-CfaN or KRAB-ZF21.16N-CfaN in the same cell
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=====Boolean logic gates via split zinc finger-based transcription factors=====
 
Lohmueller JJ et al have demonstrated the split ZF-TF reconstitution process.
 
Lohmueller JJ et al have demonstrated the split ZF-TF reconstitution process.
  
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[[File:ZF-TF.jpg|none|400px|thumb|Lohmueller JJ et al demonstrated:''After expression, the two split ZF-intein fragments bind together and undergo protein splicing to cleave away intein fragments and reconstitute the full ZF activator leading to activation of the BCR_ABL reporter.'']]
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[[File:ZF-TF.jpg|none|400px|thumb|Lohmueller JJ et al demonstrated: ''After expression, the two split ZF-intein fragments bind together and undergo protein splicing to cleave away intein fragments and reconstitute the full ZF activator leading to activation of the BCR_ABL reporter.'']]
  
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[[File:zfAND.jpg|none|400px|thumb|Lohmueller JJ et al demonstrated:''For AND gates, a ZF activator is spliced and the logical operation is computed as TRUE only when both input signals are present. For the response data shown BCR_ABL-1:GCN4 activator split fragments were used and the response promoter contains 6 copies of the BCR_ABL target site. CFP expression was measured by flow cytometry and expressed as fold change over an off-target expression control.'']]
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[[File:zfAND.jpg|none|400px|thumb|Lohmueller JJ et al demonstrated: ''For AND gates, a ZF activator is spliced and the logical operation is computed as TRUE only when both input signals are present. For the response data shown BCR_ABL-1:GCN4 activator split fragments were used and the response promoter contains 6 copies of the BCR_ABL target site. CFP expression was measured by flow cytometry and expressed as fold change over an off-target expression control.'']]
  
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[[File:zfNAND.jpeg|none|400px|thumb|Lohmueller JJ et al demonstrated:''For NAND gates, the computational module splices a ZF repressor, and the logical operation is computed as TRUE as long as both inputs are not present together. For the response data shown BCR_ABL-1:GCN4 repressor split fragments were used and the response promoter contains 6 copies of the BCR_ABL target site. CFP expression was measured by flow cytometry and expressed as fold change over an off-target expression control.'']]
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[[File:zfNAND.jpeg|none|400px|thumb|Lohmueller JJ et al demonstrated: ''For NAND gates, the computational module splices a ZF repressor, and the logical operation is computed as TRUE as long as both inputs are not present together. For the response data shown BCR_ABL-1:GCN4 repressor split fragments were used and the response promoter contains 6 copies of the BCR_ABL target site. CFP expression was measured by flow cytometry and expressed as fold change over an off-target expression control.'']]
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===coexpressed with VP64-ZF21.16N-CfaN or KRAB-ZF21.16N-CfaN in the same cell===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2549038 SequenceAndFeatures</partinfo>
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2549038 parameters</partinfo>
 
<partinfo>BBa_K2549038 parameters</partinfo>
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===References===
 
===References===

Revision as of 13:46, 12 October 2018


CfaC-ZF21.16C-NLS

This part is one of the downstream elements of our amplifier. It is constructed by fusing CfaC (Part:BBa_K2549010), ZF21.16C (Part:BBa_K2549012) and NLS (Part:BBa_K2549054), from N terminal to C terminal. CfaC is the C-terminal fragment of Cfa which is a consensus sequence from an alignment of 73 naturally occurring DnaE inteins that are predicted to have fast splicing rates. ZF21.16C is the C-terminal fragment of the zinc finger whose recognition helices for three-finger arrays are substituted by the reported synthetic zinc finger 21.16 residues on the basis of the BCR_ABL-1 artificial zinc finger[1]. NLS is a short nuclear location sequence from SV40 large T antigen. When coexpressed with VP64-ZF21.16N-CfaN (Part:BBa_K2549036) in the same cell, both fusions are formed and a transcription activating function is executed. Also, when coexpressed with KRAB-ZF21.16N-CfaN (Part:BBa_K2549037) in the same cell, both fusions are formed and a transcription repressing function is executed.

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 91
    Illegal SapI.rc site found at 21


Biology

It works as we designed

coexpressed with VP64-ZF21.16N-CfaN or KRAB-ZF21.16N-CfaN in the same cell

Boolean logic gates via split zinc finger-based transcription factors

Lohmueller JJ et al have demonstrated the split ZF-TF reconstitution process.

Lohmueller JJ et al demonstrated: After expression, the two split ZF-intein fragments bind together and undergo protein splicing to cleave away intein fragments and reconstitute the full ZF activator leading to activation of the BCR_ABL reporter.
Lohmueller JJ et al demonstrated: For AND gates, a ZF activator is spliced and the logical operation is computed as TRUE only when both input signals are present. For the response data shown BCR_ABL-1:GCN4 activator split fragments were used and the response promoter contains 6 copies of the BCR_ABL target site. CFP expression was measured by flow cytometry and expressed as fold change over an off-target expression control.
Lohmueller JJ et al demonstrated: For NAND gates, the computational module splices a ZF repressor, and the logical operation is computed as TRUE as long as both inputs are not present together. For the response data shown BCR_ABL-1:GCN4 repressor split fragments were used and the response promoter contains 6 copies of the BCR_ABL target site. CFP expression was measured by flow cytometry and expressed as fold change over an off-target expression control.


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