Part:BBa_K2446030
SV40_8_ZF_21-16 promoter
This part (Sv40 8* ZF_20-16 ) is a mammalian synthetic promoter (SynPro) based on Sv40 promoter (BBa_K2446052) and eight repeats of Zinc Finger_20-16 binding sites . We inset the 8* ZF_20-16 binding sites behind the Sv40 promoter. Sv40 promoter is a constitutive expression promoter from the simian vacuolating virus 40. In this way, we can use our mammalian synthetic transcription factors (SynTFs) which is a fusing protein ZF_20-16 _DBD_(G4S) linker_NLS_KRAB. The ZF_20-16-DBD can bind to the 8* ZF_20-16 binding sites specifically[1] and the KRAB domain can repress the expression of Sv40 8* ZF_20-16 promoter [2]. The corresponding SynTF of Sv40-8*ZF_20-16 is ZF_20-16_KRAB (BBa_K2446039).
The iGEM-team [http://2018.igem.org/Team:Fudan/Improve Fudan 2018] has utilized this part in constructing their Combiner of transmembrane binary logic gates in eukaryotic cells. Finding that placing the responsive elements downstream the promotor may have an impact on basal expression of the promotor, they adjusted the position of responsive elements to the upstream of the promotor. Furthermore, the CMV promotor has a higher level of constitutive expression, they optimized this part by changing SV40 promotor to CMV promotor. Refer to Part:BBa_K2549029 for more details.
Figure 1:(A)Design of SynTF-SynPros. (B) The specific binding sites sequences of Zinc finger we used
The information of other SynTF-SynPros is 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Experiments
SynTF-SynPro Pairs
Figure 2: the testing circuits of ZF_21-16-KRAB& Sv40-8_ ZF_21-16 pair
To make sure the SynTF-SynPro pairs work in mammalian cells, we use the circuits above to test if the ZF_21-16-KRAB can repress the expression of Sv40-8_ZF_21-16 indeed. ZF_21-16-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_21-16). These circuits are both inserted in to the mammalian expression vactor pML2. We transfect pML2-Sv40-8_ ZF_21-16 into Hela cells and measure the fluorescence intensity of mCherry by flow cytometer to get the basic expression intensity of Sv40_ ZF_21-16. We also co-transfect the pML2- ZF_21-16-KRAB with pML2-Sv40-8_ ZF_21-16 into Hela cells at the same time. Then measure the fluorescence intensity of mCherry again to get the expression intensity of Sv40-8_ZF_21-16 influenced by ZF_21-16-KRAB. The results of the experiment is showed below. The SynTF ZF_21-16 can silence the expression of the SynPro Sv40-8_ZF_21-16 in 26 folds.
Figure 3:The results of ZF_21-16-KRAB&SV40_ ZF_21-16 testing: (A) The red points is the cells before co-transfecting ZF_21-16-KRAB and the blue points is the cells after co-transfecting ZF_21-16-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_21-16-KRAB;(B) The red area is the fluorescence intensity of mCherry before co-transfecting ZF_21-16-KRAB and the blue area is the intensity after co-transfecting ZF_21-16-KRAB.(C) The statistical result of all of the SynTFs-SynPros pairs: ZF_21-16-KRAB can silence the expression intensity of Sv40-8_ ZF_21-16 in 26 folds
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.
References
[1] J. J. Lohmueller, T. Z. Armel, P. A. Silver, A tunable zinc finger-based framework for Boolean logic computation in mammalian cells. Nucleic Acids Research 40, 5180--5187 (2012).
[2] R. Witzgall, E. O'Leary, A. Leaf, D. Onaldi, J. V. Bonventre, 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).| None |