Part:BBa_K1688013

Nah7 - Upper naphthalene breakdown pathway (inc RBS, J23110 promoter)

Upper naphthalene breakdown pathway from the Nah7 plasmid of Pseudomonas putida + BBa_J23110 promotor and B0034 RBS. The operon consists of ten genes (NahAa, NahAb, NahAc, NahAd, NahB, NahC, NahD, NahE, NahF, NahQ) which in turn codes for seven enzymes that catalyse the breakdown of naphthalene into salicylate.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1927
  Illegal PstI site found at 5922
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 4182
  Illegal PstI site found at 1927
  Illegal PstI site found at 5922
  Illegal NotI site found at 8225
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 5275
  Illegal BamHI site found at 3260
  Illegal BamHI site found at 5962
  Illegal XhoI site found at 1371
  Illegal XhoI site found at 3759
  Illegal XhoI site found at 3911
  Illegal XhoI site found at 9413
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1927
  Illegal PstI site found at 5922
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1927
  Illegal PstI site found at 5922
  Illegal NgoMIV site found at 1864
  Illegal NgoMIV site found at 3639
  Illegal NgoMIV site found at 5513
  Illegal NgoMIV site found at 6125
  Illegal NgoMIV site found at 9438
  Illegal AgeI site found at 702
  Illegal AgeI site found at 3633
  Illegal AgeI site found at 6341
  Illegal AgeI site found at 8336
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2981
  Illegal BsaI site found at 3654
  Illegal BsaI site found at 4245
  Illegal BsaI site found at 5411
  Illegal BsaI.rc site found at 2815
  Illegal BsaI.rc site found at 5565
  Illegal BsaI.rc site found at 7850
  Illegal SapI.rc site found at 3345
  Illegal SapI.rc site found at 3891
  Illegal SapI.rc site found at 7742
  

Usage and Biology

Cultures with naphthalene

The upper naphthalene pathway was introduced into both DH5α and BL21 strains of E.coli, where DH5α is a cloning strain and BL21 is a strain optimized for protein expression. In the DH5α cells a medium strength promoter was used to put less strain on the cells, whereas in BL21 a strong promoter could be used to increase the expression level of the desired enzymes.

Both strains were grown in liquid culture with either no naphthalene, naphthalene directly supplied to the medium, or with naphthalene supplied in gas form as shown in figure 5. The OD of the cultures were measured after 24 and 48 hours at both OD600 (for cell growth) and for OD303 (for the presence of salicylate). The graphs in figures 6 to 11 show the experimental values obtained by spectrometry.

All the cultures containing naphthalene supplied directly to the medium showed a clear trend of significantly lower growth rates in the negative control compared to in the cells containing the pathway. This is to be expected as naphthalene is toxic to the cells and the negative control is unable to degrade it. After 24 hours, the BL21 had grown substantially more than both the negative control and the DH5α cells. This is not surprising as this strain is better at producing the enzymes of our pathway, and thus should be better at degrading naphthalene. However, after 48 hours the DH5α culture had reached similar levels of optical density as the stabilized BL21 cultures. A plausible reason is that the strain still has the pathway, though the level of expression is lower than in BL21.

The naphthalene supplied in gas form does not appear to affect the cell growth at all. However, in cultures without naphthalene the negative control grew somewhat better than cells with the construct. The reason is probably that the negative control does not have to maintain a large unnecessary plasmid.

The presence of salicylate both directly in the culture and with the cells removed, was measured at OD303. Similar results were observed at 24 hours as in above described experiment, with higher salicylate levels in BL21 compared to DH5α and the negative control. Once again, after 48 hours DH5α had approximately as high levels of salicylate as BL21. Levels of salicylate, however, appear to be far higher in cultures without naphthalene or with naphthalene in gas form, disagreeing with our original hypothesis. This may be due to interfering cells or substances. Regardless, results still show a clear trend both in salicylate levels and in cell growth, indicating that our construct is indeed being expressed and is degrading naphthalene to salicylate.