IGEM:Paris Bettencourt 2012/Notebooks/RE group/Promoter Candidates

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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.
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.
 +
This part is not available in the registry. We need to synthesize it.
* <b>Regulation:</b>
* <b>Regulation:</b>
** Induced by: L-Rhamnose  
** Induced by: L-Rhamnose  

Revision as of 11:27, 2 July 2012

Our main objective is to find inducible promoters that are not leaky.

  • Plasmid:
    • Low to medium copy BioBrick standard vector: pSB3K3


Contents

pBad

  • Regulation:
    • Induced by:Arabinose
    • Inhibited by: Glusose
  • Sequence:
    1. Overview of pBad Promoter Family: [1]
    2. pBad Part:BBa_I13453
      pBad promoter from I0500 without AraC.
    3. Inducible pBad/araC promoter:BBa_I0500 [Registry Star]
  • References:
    1. «In Vivo Induction Kinetics of the Arabinose Promoters in Escherichia coli» Casonya M. Johnson And Robert F. Schleif* Read It

pLac

  • Regulation:
    • Induced by: Lactose, IPTG
    • Repressed by: LacI
  • Sequence:
    1. 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.
    2. 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.
    3. 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.
    4. LacI Promoter Variant #4: BBa_K611024
      A mutant of the BBa_R0010 part. Lower leakage, but also lower expression.
  • References:
    1. «Tightly regulated vectors for the cloning and expression of toxic genes» Larry C. Anthony*, Hideki Suzuki, Marcin Filutowicz Read It

pTet

  • Regulation:
    • Induced by: aTc
    • Repressed by: tetR
  • Sequence:
    1. 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.
    2. 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.
  • References:
    1. «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

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.

This part is not available in the registry. We need to synthesize it.

  • Regulation:
    • Induced by: L-Rhamnose
    • Inhibited by: D-Glucose
  • Sequence:
    1. 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).
  • References:
    1. «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
    2. «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
    3. «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

Interesting links

  • Promoter measurment protocol from Imperial 2009 Read It
  • Rhamnose-inducible promoter that drives expression of I-SceI endonuclease [2]
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