Coding

Part:BBa_K2549037

Designed by: Rongrong Du   Group: iGEM18_Fudan   (2018-10-09)


KRAB-ZF21.16N-CfaN

This part is one of the downstream elements of our amplifier. It was constructed by fusing KRAB (Part:BBa_K2549055), G4S linker (Part:BBa_K2549053), ZF21.16N (Part:BBa_K2549011) and CfaN (Part:BBa_K2549009), from N terminal to C terminal. KRAB is a strong transcription repressor. 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 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
    COMPATIBLE WITH RFC[1000]


Biology

Our characterization
CfaN intein-based NAND gate. A degradable EGFP (d2EGFP) is produced downstream the promoter of the Combiner to indicate the output (CusP) strength. DBD, DNA binding domain which is zinc finger in our assay. SD, silencing-form transcriptional domain, was KRAB. RE, responsive elements. RFI, relative fluorescence intensity (comparing before and after activation). More details please visit http://2018.igem.org/Team:Fudan/Results and http://2018.igem.org/Team:Fudan/Measurement .

We show that when KRAB-ZF21.16N-CfaN co-expressed with CfaC-ZF21.16C-NLS, the expression level of d2EGFP is relatively turned down due to the formation of KRAB-ZF21.16 after auto-plicing and ligation.

The signal-noise-ratio is ok when both present. However, we are not sure why CfaC-ZF21.16C-NLS alone has effect. We have formed a couple of hypothesis and in the process of optimizing this gate. 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 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
[edit]
Categories
//cds/transcriptionalregulator/repressor
//proteindomain/binding
//proteindomain/internal/special
Parameters
None