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Progress
| Cloning | Transfection | Dox | Cytometry | Data Analyis |
|---|---|---|---|---|
| LRs transformed |
Procedure
TRE | |||||
| LacI | |||||
| Cumate | Cumate | ||||
| EGSH | Pronesterone A |
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 and pLac.
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 |
References
Ecsydone responsive promoter:
http://www.biotechniques.com/multimedia/archive/00011/01312rr03_11591a.pdf