Coding
ZF21.16C

Part:BBa_K2549012

Designed by: Rongrong Du   Group: iGEM18_Fudan   (2018-10-05)

ZF21.16 split C

This part is the C-terminal fragment of the zinc finger. Amino acid residues of the recognition helices for three-finger arrays are substituted by the reported synthetic zinc finger 21.16 residues[1] on the basis of the BCR_ABL-1 artificial zinc finger. Splicing site between the 48 and 49 residues is chosen as is reported to function the second highest among the tested 12 splicing sites[2]. This site is chosen as the +1 position residue for the CfaC (Part:BBa_K2549010) has to be a cysteine[3]. It was fused to the CfaC (Part:BBa_K2549010) intein. When (ZF-N + CfaN Part:BBa_K2549009) co-expressed with (CfaC Part:BBa_K2549010 + ZF-C) the DNA binding activity of the ZF will be restored.

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]


History

We chose ZF21.16 to split because our previous extensive characterization[4], which is summarized blew. The related parts we used previous are Part:BBa_K2446030 (containing 8 copies DNA binding sequences as Part:BBa_K2446015) and Part:BBa_K2446039 (but without the KRAB transcriptional inhibitor).

ZF21.16 binding sequence

In our 2017 Swords project: The critical step to find optional SynTF group is to find enough differently specific and orthogonal DNA binding domains. We applied two approaches to achieve this. Firstly, we widely investigated those commonly used DNA binding domain originating from different species. Secondly, we devised a platform based on artificial zinc-finger (ZF). For the first idea, we chose Gal4DBD, PIP, ZFHD1 from a large number of candidates. For the second idea, we utilized a modified 3-tendem Cys2-His2 ZF as protein chassis. By replacing the DNA-interactional amino residues on ZF modules, we can generate response-element-specific mammalian synthetic ZF (SynZF).

After our characterization, ZF43.8 and ZF21.16 stand out. They are our favorites.

Tuning the silencing fold by adjusting the response-element repeats of 2*, 4*, 8* on SynPro(S)-43-8. Values were measured by FACS recording single cell reporter fluorescence. At least 20,000 cells were analyzed for each condition. Values are presented as the mean of n = 3 ± SEM. ****, p < 0.0001.
Orthogonality of SynTF-SynPro pairs. Grids in blue rectangle showed that SynTF-SynPro pairs constructed by using SynZF as DNA binding domain with good orthogonality. Grids in pink rectangles replaced our favorite SynTF-SynPro pairs. At least 20,000 cells were analyzed for each condition in each grid in the heat map. Data were recorded by FACS at 24h after co-transfecting.
The SynTF-SynPro pairs we wired and test in our 2017 Swords project. We have further validated the ones used this year, after switching the eukaryotic expressing vector.

Note: Part:BBa_K2549011 and Part:BBa_K2549012 have the same Biology section.


References

  1. ↑ A synthetic biology framework for programming eukaryotic transcription functions. Khalil AS, Lu TK, Bashor CJ, ..., Joung JK, Collins JJ. Cell, 2012 Aug;150(3):647-58 PMID: 22863014; DOI: 10.1016/j.cell.2012.05.045
  2. ↑ 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
  3. ↑ A promiscuous split intein with expanded protein engineering applications. Stevens AJ, Sekar G, Shah NH, ..., Cowburn D, Muir TW. Proc Natl Acad Sci U S A, 2017 Aug;114(32):8538-8543 PMID: 28739907; DOI: 10.1073/pnas.1701083114
  4. ↑ http://2017.igem.org/Team:Fudan/Part_Collection
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