Coding
ZF21.16N

Part:BBa_K2549011

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

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.

Literature Characterization by AFCM-Egypt

The study used ZF21 antibody for detection of ZF21 by Western blot at 1 µg/mL. For immunofluorescence, start at 20 µg/mL.

(A)western blot analysis of ZF21 in 3T3 cell tissue lysate with ZF 21 antibodies in abscence of blocking peptide while (B) the same western blot analysis in presence of blocking peptide

charactrization of mathematical modeling by AFCM-Egypt

We are using mathematical modeling to simulate the effect of activation on the internal domain on the level of expression of our engineered exosomes so on activation of Zinc finger proteins (ZF21.16) which represent a part of the internal domain that leads to stimulation of the whole internal domain.

The graph shows the relation between activation of the internal domain of synthetic notch and increased level of engineered exosomes.



comparison between the types of internal domains of different receptors based on their ability for production of exosomes.
1)We modeled the kinetics of chimeric antigen receptor (CAR) to explain production of engineered exosomes from MSC, but it’s concluded that it wasn’t efficient as there’s nearly detected signal of exosomes production as its internal domain couldn’t be modified to be used in MSC

The graph shows the relation between activation of the internal domain of CAR receptor and the nearly detected level of engineered exosomes.



2)We modeled the kinetics of Receptors Activated Solely by Synthetic Ligands (RASSLs) to explain production of engineered exosomes from MSC, but it’s concluded that it wasn’t efficient as there’s low detected signal of exosomes production as its internal domain transcription factor activates a lot of pathways so it will not be specific to activate our internal circuit.

The graph shows the relation between activation of the internal domain of RASSLs receptor and the low detected level of engineered exosomes.



3)We modeled the kinetics of antibody scaffold receptor ( Example for it third generation of CAR receptors ) to explain production of engineered exosomes from MSC, but it’s concluded that it wasn’t efficient as there is no signal of exosomes production as its internal domain couldn’t be modified to be used in MSC .

The graph shows the relation between activation of the internal domain of antibody scaffold receptors and the nearly detected level of engineered exosomes.



4)We modeled the kinetics of syn-notch receptor to explain production of engineered exosomes from MSC, it’s concluded that it is efficient as there’s a high signal of exosomes production as its internal domain can be modified to be used in MSC .

The graph represents the relation between the activation of the internal domain of the syn-noth (represented as red line) and production of exosomes with specific cargo (represented as blue line) as the production of the engineered exosomes is initiated once the internal domain is activated.

Experimental Characterization by AFCM-Egypt

In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents in lane (P1) including CD8 alpha-his tag-mouse notch core-ZF21.16\VP64, and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figure.





We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure in lane (P1)

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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