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

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

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

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. We are going to use this part as we want pbad to be on the plasmid continaing the antitoxin, and AraC to be on the e.coli genome (along with the toxin).

  3. Inducible pBad/araC promoter:BBa_I0500 [Registry Star]

• Characterization:
  • Cambridge 2009 team

• 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.

  5. LacI Promoter + pL: BBa_R0011

     Part that seems to work well according to Antoine. Stronger Induction becauseof the pL. Only the promoter with no lacI

• 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.

• Caracterization:
  • EPF-Lausanne 2011 team

• 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

• General information:

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 system:

  L-rhamnose is taken up by the RhaT transport system, converted to L-rhamnulose by an isomerase RhaA and then phosphorylated by a kinase RhaB. Subsequently, the resulting rhamnulose-1-phosphate is hydrolyzed by an aldolase RhaD into dihydroxyacetone phosphate, which is metabolized in glycolysis, and L-lactaldehyde. The latter can be oxidized into lactate under aerobic conditions and be reduced into L-1,2-propanediol under unaerobic conditions.

  The genes rhaBAD are organized in one operon which is controlled by the [math]\displaystyle{ rhaP_{BAD} }[/math] promoter. This promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which is located in opposite direction of rhaBAD. If L-rhamnose is available, RhaR binds to the [math]\displaystyle{ rhaP_{RS} }[/math] promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose in turn binds to the [math]\displaystyle{ rhaP_{BAD} }[/math] and the [math]\displaystyle{ rhaP_{T} }[/math] promoter and activates the transcription of the structural genes. However, for the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the [math]\displaystyle{ rhaP_{BAD} }[/math] promoter has to be cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression.

• Regulation:
  • Induced by: L-Rhamnose
  • Inhibited by: D-Glucose

The E. coli rhaBRS locus. In the presence of Lrhamnose, RhaR activates transcription of rhaR and rhaS, resulting in an accumulation of RhaS. RhaS then acts as the L-rhamnose-dependent positive regulator of the rhaB promoter.

• Sequence:
  • Prha: Rhamnose->PoPS: BBa_K564001 (DNA planning, no experience)

    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).

  • The promoter can be PCR-amplified from the E. coli genome (strain MG1655). Two variants:
    1. Amplify the rhaB promotor and rhaR and rhaS regulatory genes (about 2000bp).
    2. Amplify only the sequence between rhaB and rhaR genes (287bp from base 4 095 472 to 4 095 758).
  • The promotor can be synthesized based on paper results:
    1. Site-specific excision vector pCRE5: complete sequence.

  ttaacac tcagataatg gtgctgaatt tcattacgac cagtctaaaa agcgcctgaa ttcgcgacct

• Questions to answer:
  1. Which sequences do we need? Read more papers about experiments.
  2. Do we need to express rhaR and rhaS?
  3. CRP-cAMP complex.

• 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]