Difference between revisions of "Part:BBa K2549038"

(→‎Boolean logic gates via split zinc finger-based transcription factors)
(→‎Biology)
Line 12: Line 12:
 
<!-- Add more about the biology of this part here -->
 
<!-- Add more about the biology of this part here -->
 
===Biology===
 
===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=====
 
=====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 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.
Line 23: Line 20:
  
 
[[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.'']]
 
[[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.'']]
 +
 +
===Characterization===
 +
====It works as we designed.====
 +
[[File:ANDNAND.png|none|480px|thumb|'''CfaC intein-based AND gate and NAND 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.''']]
 +
 +
These results were obtained using flow cytometry. As for the AND gate, when coexpressed with VP64-ZF21.16N-CfaN, the expression level of d2EGFP is relatively turned up compared to circumstances that they're not coexpressed. While for the NAND gate, when coexpressed with KRAB-ZF21.16N-CfaN, the expression of d2EGFP is relatively repressed.
 +
 +
  
  

Revision as of 12:15, 17 October 2018


CfaC-ZF21.16C-NLS

This part is one of the downstream elements of our amplifier. It was 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 will be produced and a transcription activating function will be executed. Also, when coexpressed with KRAB-ZF21.16N-CfaN (Part:BBa_K2549037) in the same cell, both fusions will be produced formed and a transcription repressing 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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 91
    Illegal SapI.rc site found at 21


Biology

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.
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.

Characterization

It works as we designed.

CfaC intein-based AND gate and NAND 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.

These results were obtained using flow cytometry. As for the AND gate, when coexpressed with VP64-ZF21.16N-CfaN, the expression level of d2EGFP is relatively turned up compared to circumstances that they're not coexpressed. While for the NAND gate, when coexpressed with KRAB-ZF21.16N-CfaN, the expression of d2EGFP is relatively repressed.



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