IGEM:Paris Bettencourt 2012/Notebooks/RE group/Promoter Candidates
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Our main objective is to find inducible promoters that are not leaky.
- Plasmid:
- Low to medium copy BioBrick standard vector: pSB3K3
pBAD
- Activity:
- Induced by: Glucose
- Inhibited by: Arabinose
- Sequence:
- Overview of pBad Promoter Family: [1]
- pBad Part:BBa_I13453
- pBad promoter from I0500 without AraC.
- Inducible pBad/araC promoter:BBa_I0500 [Registry Star]
- Characterization:
- References:
- «In Vivo Induction Kinetics of the Arabinose Promoters in Escherichia coli» Casonya M. Johnson And Robert F. Schleif* Read It
Plac
- Activity:
- Induced by: Lactose, IPTG
- Repressed by: LacI
- Sequence:
- lacI repressor from E. coli (+LVA): BBa_C0012
- Coding region for the LacI protein with an LVA degradation tail and without an RBS. LacI binds to the pLac regulator BBa_R0010 and PLlac01 hybrid regulator BBa_R0011 and inhibits transcription. IPTG (Isopropylthiogalactoside) binds to LacI and inhibits its operation, therefore promoting transcription. A rapid degradation tail (LVA) has been added to improve the switch time for High to Low performance of this part.
- LacIQ promoter:BBa_K091111
- This is a modified version of the lacI promoter that should bind the RNA polymerase more tightly than the wild-type lac promoter, which should reduce leaky transcription.
- Promoter (lacI regulated):BBa_R0010
- It is the natural promoter for the LacZYA operon. This part is an inverting regulator sensitive to LacI and CAP.
- LacI Promoter Variant #4: BBa_K611024
- A mutant of the BBa_R0010 part. Lower leakage, but also lower expression.
- lacI repressor from E. coli (+LVA): BBa_C0012
- References:
- «Tightly regulated vectors for the cloning and expression of toxic genes» Larry C. Anthony*, Hideki Suzuki, Marcin Filutowicz Read It
Ptet
- Activity:
- Induced by: aTc
- Repressed by: tetR
- Sequence:
- Ptet Part:BBa_R0040
- Sequence for pTet inverting regulator. Promoter is constitutively ON and repressed by TetR. TetR repression is inhibited by the addition of tetracycline or its analog, aTc.
- TetR Part:BBa_C0040
- Coding region for the TetR protein without the Ribosome Binding Site. Modified with an LVA tail for rapid degradation of the protein and faster fall time for the emission. TetR binds to the pTet regulator (Part:BBa_R0040). aTc (anhydrotetracycline) binds to TetR and inhibits its operation.
- Ptet Part:BBa_R0040
- Caracterization:
- References:
- «Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements» Rolf Lutz and Hermann Bujard* Read It
pRHA (P Rhamnose)
L-rhamnose-inducible promoter is capable of high-level recombinant protein expression in the presence of L-rhamnose, it is also tightly regulated in the absence of L-rhamnose by the addition of D-glucose.
- Activity:
- Induced by: L-Rhamnose
- Inhibited by: D-Glucose
- Sequence:
- Prha: Rhamnose->PoPS: BBa_K564001
- This part contains the rhamnose promoter and its two coregulators, rhaS and rhaR. This promoter is activated in the presence of L(+)-Rhamnose and inhibited by glucose due to catabolite repression. It can be used in a very analogous way to the arabinose promoter (i0500).
- Prha: Rhamnose->PoPS: BBa_K564001
- References:
- «Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system» Matthew J. Giacalone1, Angela M. Gentile2, Brian T. Lovitt2, Neil L. Berkley2, Carl W. Gunderson1, and Mark W. Surber2 Read It
- «DNA-Dependent Renaturation of an insoluble DNA binding Protein. Identification of the RhaS Binding Site at rhaBAD» Susan M.Egan and Robert F. Schleif Read It
- «Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations» Angelika Wegerer, Tianqi Sun and Josef Altenbuchner Read It.
- Note: Only need to express the PRhaBad promoter. The other genes are on the E.coli chromosome