Progress
| Cloning | Transfection | Dox | Cytometry | Data Analyis |
|---|---|---|---|---|
| Midis | 8/20 |
Procedure
TRE | Doxycycline | 0 | 0.1nM | 1nM | 10nM | 100nM | 1000nM |
| LacI | IPTG | 0 | 0.1mM | 0.5mM | 1mM | 5mM | 10mM |
| Cumate | Cumate | 0 | 0.5ug/ml | 1ug/ml | 5ug/ml | 10ug/ml | 50 ug/ml |
| Cumate | Cumate | 0 | 5uM | 10uM | 50uM | 100uM | 500 uM |
| EGSH | Ponasterone A | 0 | 0.1uM | 0.5uM | 1uM | 5uM | 10uM |
Small molecule inducers will be added 24 hours after transfection and 24 hours before cytometry as per standard procedure.
Notes:
Glucose in the media may be a problem with the lac system (glucose is a weak anti-inducer of the lac repressor). A paper found a two fold decrease in induction in cells cultured in glucose containing media compared to galactose substituted media.
Cumate Molecular Weight: 194 g/mol
50 ug --> (50*10^-6)g/194g/mol = 0.257*10^-6mol
50ug/ml = 0.257*10^-6mol/ml = 0.257*10^-3 mol/L = 0.257mM
Plate 1 (Control Plate)
Plate 2
|
Background
To determine the optimal level of expression of BCR components and Syk-TEVp, we are placing them under the control of inducible/repressible promoters. This experiment aims to characterize the activity of the promoters we are going to use: TREt, hEF1a-LacO, hEF1a-CuO, and EGSH.
The pLac system may not be ideal to modulate expression given it's quick on/off response. We looked into other inducible expression systems:
EGSH promoter: http://parts.igem.org/Part:BBa_K415507
Cumate promoter:
http://parts.igem.org/Part:BBa_K875001
RheoSwitch:
http://66.155.211.155/nebecomm/products_Intl/productE3000.asp
RheoSwitch uses a Gal4 activator and the gene to be expressed is under control of a Gal4 response element. We can't use that because we are already using gal4 transcriptional activation to mediate receptor activation response.
Approach
We are going to investigate the expression of mKate driven by each of the promoters with varying concentrations of inducer (dox) and repressor (IPTG).
Parts
| Part | Status |
|---|---|
| hEF1a:rtTA | |
| TRE:mKate | |
| pLac:mKate | |
| hEF1a:LacI | |
| hEF1a:CymR | |
| hEF1a-Cuo:mKate | |
| CMV5-CuO:mKate | |
| EGSH:mKate | |
| hEf1a:VgEcR-2A-RXR |
References
Ecsydone responsive promoter:
http://www.biotechniques.com/multimedia/archive/00011/01312rr03_11591a.pdf
Lac repression system:
Cumate induction system:
Data
pTRE + Doxycycline:
| 0 nM Dox | 0.1 nM Dox | 1 nM Dox | 10 nM Dox | 100 nM Dox | 1000 nM Dox |
|---|---|---|---|---|---|
 |  |  |  |  |  |
hEF1a-LacO + IPTG
| 0 mM IPTG | 0.1 mM IPTG | 0.5 mM IPTG | 1 mM IPTG | 5 mM IPTG | 10 mM IPTG |
|---|---|---|---|---|---|
 |  |  |  |  |  |
hEF1a-CuO + Cumate
| 0 uM Cumate | 5 uM Cumate | 10 uM Cumate | 50 uM Cumate | 100 uM Cumate | 500 uM Cumate |
|---|---|---|---|---|---|
 |  |  |  |  |  |
EGSH + Ponasterone A
| 0 uM PonA | 0.1 uM PonA | 0.5 uM PonA | 1 uM PonA | 5 uM PonA | 10 uM PonA |
|---|---|---|---|---|---|
 |  |  |  |  |  |
| *bad transfection |