Difference between revisions of "Part:BBa K2549036"

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===Biology===
 
===Biology===
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=====Our characterization=====
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[[File:AND-test.png|none|480px|thumb|'''CfaN intein-based AND gate.  A degradable EGFP (d2EGFP) is produced downstream the promoter of the Combiner to indicate the output strength. DBD, DNA binding domain which is zinc finger in our assay. AD, activating-form transcriptional domain, was VP64. RE, responsive elements. RFI, relative fluorescence intensity (comparing before and after activation).''']]
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We show that when VP64-ZF21.16N-NLS-CfaN co-expressed with CfaC-ZF21.16C-NLS, the expression level of d2EGFP is relatively turned up due to the formation of VP64-ZF21.16 after auto-plicing and ligation. The signal-noise-ratio is still not optimal, and we are improve the design by switching the split position as well as adding linkers.
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=====Boolean logic gates via split zinc finger-based transcription factors=====
 
=====Boolean logic gates via split zinc finger-based transcription factors=====
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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.'']]
 
[[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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===Characterization===
 
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=====It works as we designed =====
 
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[[File:AND-test.png|none|480px|thumb|'''CfaN intein-based AND 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. AD or SD, activating- or silencing-form transcriptional domain. RE, responsive elements. RFI, relative fluorescence intensity.''']]
 
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Flow cytometry results suggest that when coexpressed with CfaC-ZF21.16C-NLS, the expression level of d2EGFP is relatively turned up compared to none of them exists or only one of them exists.
 
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Revision as of 20:48, 17 October 2018


VP64-ZF21.16N-CfaN

This part is one of the downstream elements of our amplifier. It is constructed by fusing VP64 (Part:BBa_K2549057), G4S linker (Part:BBa_K2549053), ZF21.16N (Part:BBa_K2549011) and CfaN (Part:BBa_K2549009), from N terminal to C terminal. VP64 is a tetrameric VP16 transcription activator which shows ultrahigh transcription activation function. G4S is a glycine-rich peptide linker whose sequence is GGGGS. ZF21.16N is the N-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]. CfaN is the N-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. When coexpressed with CfaC-ZF21.16C-NLS (Part:BBa_K2549038) in the same cell, both fusions will be produced and a transcription activating function will be 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
    COMPATIBLE WITH RFC[1000]


Biology

Our characterization
CfaN intein-based AND gate. A degradable EGFP (d2EGFP) is produced downstream the promoter of the Combiner to indicate the output strength. DBD, DNA binding domain which is zinc finger in our assay. AD, activating-form transcriptional domain, was VP64. RE, responsive elements. RFI, relative fluorescence intensity (comparing before and after activation).

We show that when VP64-ZF21.16N-NLS-CfaN co-expressed with CfaC-ZF21.16C-NLS, the expression level of d2EGFP is relatively turned up due to the formation of VP64-ZF21.16 after auto-plicing and ligation. The signal-noise-ratio is still not optimal, and we are improve the design by switching the split position as well as adding linkers.


Boolean logic gates via split zinc finger-based transcription factors

Lohmueller JJ et al have demonstrated the split ZF-TF reconstitution process. Please note that we used Cfa split intein (Part:BBa_K2549009 and Part:BBa_K2549010) but not dnaB reported below.

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.


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