Coding
TF-KRAB-5

Part:BBa_K2446041

Designed by: Yijie Pan   Group: iGEM17_Fudan   (2017-10-21)


ZF_43-8_KRAB

This part is one of our mammalian synthetic transcription factors (SynTFs) based on Cys2-His2 zinc finger 43-8 (ZF_43-8) as protein chassis. [1]. ZF_43-8_KRAB (or TF-KRAB-5 in our project) containing three core domains from N-terminal to C-terminal: ZF_43-8 DNA binding domain, nuclear location sequence and KRAB transcription regulating domain [2].And a (G4S) linker was added between DBD and NLS for providing region flexibility [3]. ZF_43-8-DBD enable binding to specific DNA sequences(as figure 1 show), so that we can use ZF_43-8-KRAB as a specific transcription factors to repress the expression of our mammalian synthetic promoters SV40_2/4/8_ZF_43-8(BBa_K2446026)/(BBa_K2446027)/(BBa_K2446028). Each promoter has different repeats of ZF_43-8 binding sites. In this way, the silencing intensity of SynTF is adjustable.

Figure 1:(A)Design of SynTF-SynPros. (B) The specific binding sites sequences of Zinc finger we used

The information of other SynTF-SynPros are showed in the table below.

SynTFs SynPros
Gal4-KRAB(TF-KRAB-1) BBa_K2446037 Sv40-UAS(Sv40-UAS) BBa_K2446036
ZF_PIP_KRAB(TF-KRAB-2) BBa_K2446045 SV40_2/4/8_PIP BBa_K2446033/BBa_K2446034/BBa_K2446035
ZF_21-16KRAB(TF-KRAB-3) BBa_K2446039 SV40_8_ZF_21-16 BBa_K2446030
ZF_42-10_KRAB(TF-KRAB-4) BBa_K2446040 SV40_8_ZF_42-10 BBa_K2446025
ZF_43-8_KRAB(TF-KRAB-5) BBa_K2446041 SV40_2/4/8_ZF_43-8 BBa_K2446026/BBa_K2446027/BBa_K2446028
ZF_54-8_KRAB(TF-KRAB-6) BBa_K2446042 SV40_8_ZF_54-8 BBa_K2446029
ZFHD1_KRAB(TF-KRAB-7) BBa_K2446043 SV40_4_ZFHD1 BBa_K2446032


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 301
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 184
    Illegal AgeI site found at 19
  • 1000
    COMPATIBLE WITH RFC[1000]


Experiments

An tunable SynTF-SynPro Pair

Figure 2: the testing circuits of ZF_43-8-KRAB& Sv40-8_ ZF_43-8 pair

Figure 2: the testing circuits of ZF_43-8-KRAB& Sv40-8_ ZF_43-8 pair

This pair of SynTF-SynPro works well in mammalian cells. To make sure the SynTF-SynPro pairs work in mammalian cells, we use the circuits above to test if the ZF_43-8-KRAB can repress the expression of Sv40-8_ZF_43-8 indeed. ZF_43-8-KRAB is linked to the C terminal of EGFP by the link of P2A. And mCherry expressions is controlled by corresponding SynPro (Sv40-8_ ZF_43-8). These circuits are both inserted in to the mammalian expression vactor pML2. We transfect pML2-Sv40-8_ ZF_43-8 into Hela cells and measure the fluorescence intensity of mCherry by flow cytometer to get the basic expression intensity of Sv40_ ZF_43-8. We also co-transfect the pML2- ZF_43-8-KRAB with pML2-Sv40-8_ ZF_43-8 into Hela cells at the same time. Then measure the fluorescence intensity of mCherry again to get the expression intensity of Sv40-8_ZF_43-8 influenced by ZF_43-8-KRAB. The results of the experiment is showed below. The SynTF ZF_43-8 can silence the expression of the SynPro Sv40-8_ZF_43-8 in 23 folds.

An tunable SynTF-SynPros pair. The zinc finger is a highly structured motif. In tandem with multiple zinc finger motif, the recognition precision can be enhanced in its one part of usage in genome editing [4]. We applied this strength trying on constructing tunable SynTF-SynPro pairs. We observed that the inhibitor intensity was enhanced by adding more RE repeats. In the same way, we test the silencing intensity of c-KRAB to the Sv40_ promoter with 2/4/8 repeats of ZF_43-8 binding sites. There shows a significant difference of silencing intensity (figure 3-D), so that we can use the same way to engineer and adjust the SynPros that we need.

Figure 3:The results of ZF_43-8-KRAB&SV40_ ZF_43-8 testing: (A) The red points is the cells before co-transfecting ZF_43-8-KRAB and the blue points is the cells after co-transfecting ZF_43-8-KRAB. It’s easy to see that the red points depart from the diagonal and higher than the blue points. So the expression of mCherry silenced after the expression of ZF_43-8-KRAB;(B) The red area is the fluorescence intensity of mCherry before co-transfecting ZF_43-8-KRAB and the blue area is the intensity after co-transfecting ZF_43-8-KRAB.(C) The statistical result of all of the SynTFs-SynPros pairs: ZF_43-8-KRAB can silence the expression intensity of Sv40-8_ ZF_43-8 in 23 olds.(D) Tuning the silencing fold by adjusting the RE repeats of 2*, 4*, 8* on SynPro(S)-43-8. Values are the mean of n = 3 ± SEM. ****, p ＜ 0.0001.

Figure 3:The results of ZF_43-8-KRAB&SV40_8_ZF_43-8 testing: (A) The red points is the cells before co-transfecting ZF_43-8-KRAB and the blue points is the cells after co-transfecting ZF_43-8-KRAB. It’s easy to see that the red points depart from the diagonal and higher than the blue pints. So the expression of mCherry silenced after the expression of ZF_43-8-KRAB;(B) The red area is the fluorescence intensity of mCherry before co-transfecting ZF_43-8-KRAB and the blue area is the intensity after co-transfecting ZF_43-8-KRAB.(C) The statistical result of all of the SynTFs-SynPros pairs: ZF_43-8-KRAB can silence the expression intensity of Sv40-8_ ZF_43-8 in 23folds.(D) Tuning the silencing fold by adjusting the RE repeats of 2*, 4*, 8* on SynPro(S)-43-8. Values are the mean of n = 3 ± SEM. ****, p ＜ 0.0001.

SynTF-SynPro Orthogonality

To construct our [http://2017.igem.org/Team:Fudan/Model/GTN| Strip module], more than one SynTF-SynPro pairs would be applied. Thus, the interaction between the pairs would influence or ruin our construction. We did massive orthogonality experiments to avoid that. We observed all of the 5 pairs were actually orthogonal, as you could see the grids on the diagonal were always the darkest. The three DBDs commonly used in previous works were didn’t let us down. However, the expression level of these RE loaded SynPros were relative low compared to SynPro(S)-ZF serials. As the blue rectangle in the lower right corner of the orthogonality may showed the SynPro(S)-ZF has high basic expression with unpaired SynTFs, but could be silenced to the similar fold of commonly used DBDs corresponding SynPros. The SynPro(S)-ZF was likely won’t be target by other unpaired DBD, hence the purple appeared on the bottom rows.

Figure 4: the SynTF-SynPro pairs’ Orthogonality. Grids in blue rectangle showed that SynTF-SynPro pairs constructed by using SynZF as DBD with well orthogonality. Grids in pink rectangles replaced our favorite SynTF-SynPro pairs. At least 20,000 cells were analyzed for each condition in both histogram and each grid in heat map. Data are recorded by FACS at 24h after cotransfecting.

Figure 4: the SynTF-SynPro pairs’ Orthogonality. Grids in blue rectangle showed that SynTF-SynPro pairs constructed by using SynZF as DBD with well orthogonality. Grids in pink rectangles replaced our favorite SynTF-SynPro pairs. At least 20,000 cells were analyzed for each condition in both histogram and each grid in heat map. Data are recorded by FACS at 24h after cotransfecting.

References

[1] Lohmueller, J. J., Armel, T. Z. & Silver, P. A. A tunable zinc finger-based framework for Boolean logic computation in mammalian cells. Nucleic Acids Research 40, 5180--5187 (2012).

[2] Witzgall, R., O'Leary, E., Leaf, A., Onaldi, D. & Bonventre, J. V. The Krüppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. Proceedings of the National Academy of Sciences 91, 4514-4518 (1994).

[3] Chen, X., Zaro, J. L. & Shen, W.-C. Fusion protein linkers: property, design and functionality. Advanced drug delivery reviews 65, 1357-1369 (2013).

[4] A. Klug, The discovery of zinc fingers and their applications in gene regulation and genome manipulation. Annual review of biochemistry 79, 213--231 (2010).

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