Part:BBa_K270001

Pr XylR

PURPOSE

This part is necessary for the regulation of the Pu promoter. We created this part to test Bba_K270004, Pr XylR Pu GFP, in E. coli.

ORIGINS

Pr XylR 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 degrades toluene into catechol and the lower pathway expressed by Pm degrades catechol into pyruvate and acetaldehyde.

Pr PROMOTER

Pr is a constituative promoter that expresses the XylR regulator protien. When there is no toluene, XylR represses the Pr promoter and when there is toluene, XylR makes a complex with toluene that activates the Pu promoter expressing the upper pathway for toluene degradation. The XylR-toluene complex also relulates Ps and XylS, the regulatory promoter and protien for the lower pathway.

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

CONSTRUCTION

This part was created by using colony PCR to amplify the 41246-43570 bp region of the NCBI sequence of Pseudomonas putida plasmid pWW0. For this reason no biobrick scars appear in the part. The Pr primer is not annotated on the NCBI sequence, but we assumed it was 200 bp upstream from the start of the XylR gene. Cutsites were added to the primers to make the part biobrick compatable. The forward primer (5’ TATACTAGTCTGGGGCGAGAGGCGACGAC 3’) contained a SpeI cutsite and the reverse primer (5’ GGGGAATTCTAGAATGTGGGCTGCTTGGTG 3’) contained a EcoRI and XbaI cutsite.

For more detailed information about the Pr XylR construction see [http://2009.igem.org/Ann%27s_NotebookAnn's Notebook]

CONSIDERATIONS WHEN WORKING WITH Pr XylR

This part can only be digested with EcoRI and SpeI to insert the part in front of another part because the XylR gene contains a PstI cutsite.

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
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1064
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 887
  Illegal PstI site found at 1064
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 507
  Illegal BglII site found at 1969
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1064
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1064
  Illegal NgoMIV site found at 320
  Illegal NgoMIV site found at 673
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1160