Part:BBa_K2549011
ZF21.16 split N
This part is the N-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 CfaN (Part:BBa_K2549009) intein, and will be fused to ZF21.16 split C (Part:BBa_K2549012) once both fusions were expressed inside the same cell.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Biology
We chose ZF21.16 to split because our previous extensive characterization[4], which is summarized blew. The related two parts we used previous are Part:BBa_K2446030 (containing 8 copies DNA binding sequences as Part:BBa_K2446015) and Part:BBa_K2446039 (without the KRAB transcriptional inhibitor).
In our 2017 Swords project: The critical step to find optional SynTF group is to find enough differently specific and orthogonal DBDs. We applied two approaches to achieve this. Firstly, we widely investigated those commonly used DBD 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 RE-specific mammalian synthetic ZF (SynZF).
After our characterization, ZF43.8 and ZF21.16 stand out. They are our favorites.
References
- ↑ 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
- ↑ 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
- ↑ 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
- ↑ http://2017.igem.org/Team:Fudan/Part_Collection
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