Part:BBa_K3132105
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.
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).
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).
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).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1085
Illegal NgoMIV site found at 1220 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 475
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