Part:BBa_K270000

Pu Promoter

PURPOSE

This promoter has the ability to sensing toluene when associated with the regulating protien XylR. The goal of our project is to link the absence of toluene with cell death. This is used in our bioremediation project to clean up toluene pollution because it would help prevent the bacteria from poliferating in the environment when there is no more toluene present.

ORIGINS

The Pu promoter is found in Pseudomonas putida mt-2 on the pWW0-TOL plasmid which contains the metabolic pathway for degrading toluene.

pWW0-TOL PLASMID REGULATION

The regulation for the degration of toluene on this plasmid is shown in the figure below.

The image is adapted from Burlage, R. S., S. W. Hooper, and G. S. Sayler. 1989. The TOL (pWWO) catabolic plasmid. Appl. Environ. Microbiol. 55: 1323-1328.

Toluene is degraded through two pathways: the upper and lower, meta-pathway. The upper pathway expressed by Pu and degrades toluene into catechol. The lower pathway expressed by Pm and it degrades catechol into pyruvate and acetaldehyde.

Pu PROMOTER

When activated in P. putida mt-2, the Pu promoter expresses the upper pathway for degrading toluene which consists of XylC, XylA and XylB. One product of the upper pathway is benzoate which can then combine with XylS, the regulating protien for the lower pathway, to activate the Pm promoter to finish the toluene degradation.

The Pu promoter region is the 200 bp sequence upstream of the XylU gene. The reason for amplifing this large promoter region is to include the upstream activation sequences (UAS) (Ramos et al, 1997).

CONSTRUCTION

This part was created by using colony PCR to amplify the 75381-75720 bp region of the NCBI sequence of Pseudomonas putida plasmid pWW0. The Pu promoter is not annotated on the sequence, but we assumed it was 200 bp upstream from the start of the XylU gene. Cutsites were added to the primers to make the part biobrick compatable. The forward primer (5’GGTGCTGCAGACTAGTATTGAAGGGTCACCACTATTTTTATTTTA 3’) containes SpeI and PstI cutsites and the reverse primer (5’ ATGAATTCTCTAGACCTGCTGGAGGGCGTGAAC 3’) containes EcoRI and XbaI cutsites.

For more detailed information about the Pu promoter construction see [http://2009.igem.org/Ann%27s_NotebookAnn's Notebook] and [http://2009.igem.org/Nick%27s_NotebookNick's Notebook].

CONSIDERATIONS WHEN WORKING WITH THE Pu PROMOTER

This promoter will only work if used with the Pr XylR regulator construct.

REFERENCES

J.L. Ramos, S. Marques and K.N. Timmis. Annu. Rev. Microbiol. 51 (1997), pp. 341–373.

For more information see the [http://2009.igem.org/Team:Michigan/Project Michigan 2009 Wiki]

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 295
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]