Part:BBa_K2549036

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. 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-CfaN co-expressed with CfaC-ZF21.16C, 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 improving the design by switching the split position as well as adding linkers. Please note Part:BBa_K2549036 contains a transcriptional activation domain VP64, while Part:BBa_K2549037 has a transcriptional repression domain KRAB.

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.