Team:Uppsala/Results naph
Naphthalene pathway
Lifting of naphthalene pathway
Our concrete goals in the lab were to extract the naphthalene degrading pathway with genes NahA to NahF both with and without its native promoter through PCR. As is confirmed by colony PCR and electrophoresis, the pathway was successfully lifted from the Nah7 plasmid.
Assembly of promoter, pathway and backbone
The PCR product, namely the naphthalene pathway, was successfully assembled into a standard iGEM backbone through 3A assembly, confirmed through colony PCR, and two different promoters were added through standard assembly due to unwanted restriction sites in the pathway sequence.
Sequencing and proteomics
Due to financial and temporal limitations, no sequencing or proteomic studies could be performed.
Plates with naphthalene in lid
To assess differences in survivability between the cells containing the naphthalene degrading pathway and negative control cells containing an RFP-coding gene. Plates were split in two parts, one containing the naphthalene degrading bacteria and one with the negative control. Fixed amounts of naphthalene crystals ranging from 50 mg to 2 g were then placed in the lid of each plate, to determine the difference in growth rate. However, no visible difference was observed. These results are consistent with results from experiments with liquid cultures where naphthalene was also supplied in gas form.
Cultures with naphthalene
The upper naphthalene pathway was introduced into both DH5α and BL21DE3 strains of E.coli, where DH5α is a cloning strain and BL21DE3 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 BL21DE3 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 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 BL21DE3 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 BL21DE3 cultures. A plausible reason is that the strain still has the pathway, though the level of expression is lower than in BL21DE3.
The naphthalene supplied in gas form did 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 did 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 BL21DE3 compared to DH5α and the negative control. After 48 hours DH5α had approximately as high levels of salicylate as BL21DE3. 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.