Part:BBa_K2549038

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. 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.