Difference between revisions of "Part:BBa K3132105"
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<partinfo>BBa_K3132105 short</partinfo>
 
<partinfo>BBa_K3132105 short</partinfo>
  
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This part is transformed based on the part: PIP_KRAB(BBa_K2446038) of iGEM17_Fudan.The part is one of our synthetic transcription factors (SynTFs) based on 2 parts, PIP-KRAB and Gal4-VP64(BBa_K3132000).Its parental form PIP-KRAB, which was developed by iGEM_Fudan, serves as an inhibitory transcription factor because of the KRAB domain; while the added part Gal4-VP64, an improved part of our team this year, serves as an activating transcription factor because both of the Gal4 and VP64 domain.This is why we call it “bifunctional transcription factor”. PIP-KRAB-Gal4-VP64 contains six core domains from N-terminal to C-terminal: PIP DNA binding domain, nuclear location sequence, KRAB transcription regulating domain, GAL4 DNA binding domain, another nuclear location sequence and VP64 transcription regulating domain[1]. And 2 (G4S) linkers were added between DBD and NLS for providing region flexibility[2]. PIP-DBD and Gal4-DBD enable binding to specific DNA sequences, together with KRAB and VP64 constitute the inhibitory and activating part, respectively. So that we can use it as a bifunctional transcription factor to either activate or repress the expression of our downstream synthetic promoter elements and minimalCMV when binding to specific DNA sequences .
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==PIP-KRAB-Gal4-VP64, a bifunctional transcription factor==
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This part is a bifunctional transcription factor improved based on PIP-KRAB (BBa_K2446038) and Gal4-VP64 (BBa_K3132000). PIP-KARB is an inhibitory transcription factor (TF) submitted by team Fudan in 2017, whereas Gal4-VP64 is an activating TF submitted by SMMU-China in 2019. Here, we combined these two parts into one complex, so that it can inhibit gene controlled by PIR-CMV promoter (BBa_K3132101) and meanwhile activate UAS-minCMV promoter (BBa_K3132100). This part is useful when a user wants to specifically implement inhibitory and activating regulation upon two different genes at the same time. Because of this function, we called it a bifunctional transcription factor.
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[[File:T--SMMU-China--FuseTF 1.png|800px|thumb|center]]
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<b>Design</b>
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When designing genetic circuits, we frequently want to inhibit one gene while activate another gene at the same time. To realize this goal, we combined PIP-KRAB and Gal4-VP64 with a (GGGGS)3 linker (Figure. 1a). PIP-KRAB could bind inhibit 8*PIR-CMV promoter  (BBa_K3132101) and Gal4-VP64 could activate UAS-minCMV promoter (BBa_K3132100).
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==Characterization==
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To characterize this part, we first examined its inhibitory and activating activity separately and compared with its parental part PIP-KRAB. Next, we tested and verified the two functions in the same cells.
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<b>Experiment 1: Inhibitory activity of bifunctional TF and comparison with PIP-KRAB</b>
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To demonstrate that bifunctional TF has inhibitory effect and is comparable to that of PIP-KRAB, we transfected them with mCherry reporter plasmid into HEK293 cells. Both TFs was connected to a P2A-eGFP gene, so that we could eliminate bias in expression level by normalizing with eGFP fluorescence intensity. The results suggested both bifunctional TF and PIP-KRAB had a strong activity (Figure. 1).
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[[File:T--SMMU-China--FuseTF 2.png|600px|thumb|center|Figure. 1 Inhibitory activity of bifunctional TF and PIP-KRAB.]]
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<b>Experiment 2: Activating effect of bifunctional TF</b>
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The bifunctional TF plasmid was co-transfected with UAS-minCMV promoter-mCherry reporter plasmid. Fluorescence intensity was measured 24h later, and the bifunctional TF showed robust activating effect (Figure. 2).
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[[File:T--SMMU-China--FuseTF 3.png|600px|thumb|center|Figure. 2 Activating effect of bifunctional TF.]]
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<b>Experiment 3: Two functions in the same cell</b>
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After verifying the two functions independently, we next co-transfected bifunctional TF, 8*PIR-CMV promoter-eGFP, and UAS-minCMV promoter-mCherry into HEK293 cells. We anticipated that compared with control group which shows green light and no red light, the experiment group should on the contrary shows red light and no red light. The results were consistent with our speculation and only a hint of green light could be seen in bifunctional TF group (Figure. 3).
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[[File:T--SMMU-China--FuseTF 4.png|1100px|thumb|center|Figure. 3 Activating and inhibiting effect of bifunctional TF in the same cell.]]
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Revision as of 11:36, 20 October 2019


PIP-KRAB-Gal4-VP64

PIP-KRAB-Gal4-VP64, a bifunctional transcription factor

This part is a bifunctional transcription factor improved based on PIP-KRAB (BBa_K2446038) and Gal4-VP64 (BBa_K3132000). PIP-KARB is an inhibitory transcription factor (TF) submitted by team Fudan in 2017, whereas Gal4-VP64 is an activating TF submitted by SMMU-China in 2019. Here, we combined these two parts into one complex, so that it can inhibit gene controlled by PIR-CMV promoter (BBa_K3132101) and meanwhile activate UAS-minCMV promoter (BBa_K3132100). This part is useful when a user wants to specifically implement inhibitory and activating regulation upon two different genes at the same time. Because of this function, we called it a bifunctional transcription factor.

T--SMMU-China--FuseTF 1.png

Design When designing genetic circuits, we frequently want to inhibit one gene while activate another gene at the same time. To realize this goal, we combined PIP-KRAB and Gal4-VP64 with a (GGGGS)3 linker (Figure. 1a). PIP-KRAB could bind inhibit 8*PIR-CMV promoter (BBa_K3132101) and Gal4-VP64 could activate UAS-minCMV promoter (BBa_K3132100).

Characterization

To characterize this part, we first examined its inhibitory and activating activity separately and compared with its parental part PIP-KRAB. Next, we tested and verified the two functions in the same cells. Experiment 1: Inhibitory activity of bifunctional TF and comparison with PIP-KRAB To demonstrate that bifunctional TF has inhibitory effect and is comparable to that of PIP-KRAB, we transfected them with mCherry reporter plasmid into HEK293 cells. Both TFs was connected to a P2A-eGFP gene, so that we could eliminate bias in expression level by normalizing with eGFP fluorescence intensity. The results suggested both bifunctional TF and PIP-KRAB had a strong activity (Figure. 1).

Figure. 1 Inhibitory activity of bifunctional TF and PIP-KRAB.

Experiment 2: Activating effect of bifunctional TF The bifunctional TF plasmid was co-transfected with UAS-minCMV promoter-mCherry reporter plasmid. Fluorescence intensity was measured 24h later, and the bifunctional TF showed robust activating effect (Figure. 2).

Figure. 2 Activating effect of bifunctional TF.

Experiment 3: Two functions in the same cell After verifying the two functions independently, we next co-transfected bifunctional TF, 8*PIR-CMV promoter-eGFP, and UAS-minCMV promoter-mCherry into HEK293 cells. We anticipated that compared with control group which shows green light and no red light, the experiment group should on the contrary shows red light and no red light. The results were consistent with our speculation and only a hint of green light could be seen in bifunctional TF group (Figure. 3).

Figure. 3 Activating and inhibiting effect of bifunctional TF in the same cell.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1085
    Illegal NgoMIV site found at 1220
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 475