Team:Fudan/Part Collection
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As a team focusing on real-world problem, we found it is hard to obtain sufficient mammalian Biobricks on the Registry of Standard Biological Parts comparing to the convenience for project in bacteria. In synthetic biology, one central route is to construct a controllable biological network, and electing suitable transcription factor and corresponding promoter is vital.
The best way to predict the future is to invent it. As we couldn’t find Biobricks that were completely satisfying to us, we just invent it.
For this year’s iGEM competition, we present an approach to design customized mammalian synthetic transcription factor (SynTF) - synthetic promoter (SynPro) pairs. This set is a powerful toolbox to construct customized and orthogonal transcriptional network.
SynTFs enable binding to user-specified DNA sequences, response element (REs), on SynPros then silencing or activating their transcription. The SynTFs we designed were in an unified style containing three core domains from N-terminal to C-terminal: DNA binding domain (DBD), nuclear location sequence (NLS), transcription regulating domain. We chose (G4S) as linker to be added between DBD and SV40 NLS for region flexibility. We could choose KRAB or VP64 as transcription regulating domain to construct silencing- or activating-form SynTFs (SynTF(S)s and SynTF(A)s). The structures of their corresponding silencing- or activating-form SynPros (SynPro(S)s and SynPro(A)s) were pSV40-N*RE or N*RE-minCMV.
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).
1. It is a unified design for applying both natural DBDs and artificial SynZFs as DBDs on SynTFs.
2. It is a tunable design as you can adjust the silencing or activating fold by different repeats of REs.
3. It is a universal design because ZF is a highly modular motif. You can design more than 49 specific SynZF–RE pairs theoretically.
Cause the following reasons, actually functional SynZFs are much less than 49:
1. one unit SynZF motif cannot recognize all the permutation of 3 consecutive bases.
2. SynTF are probably not all complete orthogonal to each other.
3. RE repeats on SynPro can influence the function of chassis promoters.
Thus, before you applying our idea into your project. A pilot test is strongly needed.
Integrated information about Part Collection posted on Demonstrate: Wiring orthogonal and tunable SynTF-SynPro repertoire. You cannot miss it.
We listed Biobricks belong to Part Collection 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_PIP | (BBa_K2446033) |
| SV40_4_PIP | (BBa_K2446034) | ||
| SV40_8_PIP | (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_ZF_43-8 | (BBa_K2446026) |
| SV40_4_ZF_43-8 | (BBa_K2446027) | ||
| SV40_8_ZF_43-8 | (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) |
