| BACE1 circuit | miRNA circuit | Expected Result |
|---|---|---|
| Hef1a/BACE1-eYFP | Hef1a/miRNA-GFP | No BACE1 expression (positive control) |
| Hef1a/BACE1-eYFP | no miRNA | BACE1 expression (negative control) |
| Hef1a/BACE1-eYFP | TRE-t/miRNA-GFP + Dox | No BACE1 expression |
| Hef1a/BACE1-eYFP | TRE-t/miRNA-GFP + no Dox | BACE1 expression |
Step 1: Demonstrate constitutive promotion of BACE1 (N Terminal eYFP fusion)
| Protocol | Parts | Done? |
|---|---|---|
| PCR | eYFP, BACE1 | |
| GG | eYFP PCRP, BACE1 PCRP, GGDonr | |
| LR | pENTR_Hef1a, pENTR_BACE1_eYFP, pDest | |
| Transform | ||
| Purify | ||
| Transfect | ||
| Assay | ||
| Image |
Step 1.5 : Demonstrate constitutive promotion of BACE1 (C Terminal eYFP fusion)
| Protocol | Parts | Done? |
|---|---|---|
| PCR | eYFP, BACE1 | |
| GG | eYFP PCRP, BACE1 PCRP, GGDonr | |
| LR | pENTR_Hef1a, pENTR_BACE1_eYFP, pDest | |
| Transform | ||
| Purify | ||
| Transfect | ||
| Assay | ||
| Image |
|
Positive Control |
| |||
0 nM Dox |
0 nM Dox |
1 nM Dox |
1 nM Dox |
10 nM Dox |
10 nM Dox |
100 nM Dox |
100 nM Dox |
1000 nM Dox |
1000 nM Dox |
2000 nM Dox |
2000 nM Dox |